Custom-made meganuclease and use thereof

ABSTRACT

New rare-cutting endonucleases, also called custom-made meganucleases, which recognize and cleave a specific nucleotide sequence, derived polynucleotide sequences, recombinant vector cell, animal, or plant comprising said polynucleotide sequences, process for producing said rare-cutting endonucleases and any use thereof, more particularly, for genetic engineering, antiviral therapy and gene therapy.

This application is a continuation of U.S. application Ser. No.10/543,556, which is the US national phase of International ApplicationNo. PCT/IB2004/000827, filed on Jan. 28, 2004, which designated the U.S.and claims the benefit of U.S. Provisional Application Nos. 60/442,911and 60/491,535, filed on Jan. 28, 2003, and on Aug. 1, 2003,respectively. Each of the above applications is hereby incorporated byreference in its entirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 23, 2012, isnamed SEQUENCE LISTING 15471US2.txt and is approximately 12,621 bytes insize.

The present invention relates to new rare-cutting endonucleases, alsocalled custom-made meganucleases, which recognize and cleave a specificnucleotide sequence, to polynucleotide sequences encoding for said newrare-cutting endonucleases, to a vector comprising one of saidpolynucleotide sequences, to a cell, an animal, or a plant comprisingone of said polynucleotide sequences or said rare-cutting endonucleases,to a process for producing one of said rare-cutting endonucleases andany use of the disclosed products and methods. More particularly, thisinvention contemplates any use such rare-cutting endonuclease forgenetic engineering, antiviral therapy, genome therapy and gene therapy.

Homing endonucleases constitute a family of very rare-cuttingendonucleases. It was first characterised at beginning of the Ninetiesby the use (in vivo) of the protein 1-Sce I (Omega nuclease encoded by amitochondrial group I intron of the yeast Saccharomyces cerevisiae).Homing endonucleases encoded by introns ORF, independent genes orintervening sequences (inteins) present striking structural andfunctional properties that distinguish them from “classical” restrictionenzymes (generally from bacterial system R/MII). They have recognitionsequences that span 12-40 bp of DNA, whereas “classical” restrictionenzymes recognise much shorter stretches of DNA, in the 3-8 bp range (upto 12 bp for rare-cutter). Therefore, the homing endonucleases present avery low frequency of cleavage, even in the human genome.

Furthermore, general asymmetry of homing endonuclease target sequencescontrasts with the characteristic dyad symmetry of most restrictionenzyme recognition sites. Several homing endonucleases encoded byintrons ORF or inteins have been shown to promote the homing of theirrespective genetic elements into allelic intronless or inteinless sites.By making a site-specific double-strand break in the intronless orinteinless alleles, these nucleases create recombinogenic ends, whichengage in a gene conversion process that duplicates the coding sequenceand leads to the insertion of an intron or an intervening sequence atthe DNA level.

Homing endonucleases fall into 4 separated families on the basis ofpretty well conserved amino acids motifs. For review, see Chevalier andStoddard (2001, Nucleic Acids Research, 29, 3757-3774). One of them isthe dodecapeptide family (dodecamer, DOD, D1-D2, LAGLI-DADG, P1-P2).This is the largest family of proteins clustered by their most generalconserved sequence motif: one or two copies (vast majority) of atwelve-residue sequence: the di-dodecapeptide. Homing endonucleases withone dodecapetide (D) are around 20 kDa in molecular mass and act ashomodimer. Those with two copies (DD) range from 25 kDa (230 AA) to 50kDa (HO, 545 AA) with 70 to 150 residues between each motif and act asmonomer. Cleavage is inside the recognition site, leaving 4 nt staggeredcut with 3′OH overhangs. I-Ceu I and I-Cre I illustrate the homingendonucleases with one Dodecapeptide (mono dodecapeptide). I-Dmo I,I-Sce I, PI-Pfu I and PI-Sce I illustrate homing endonucleases with twoDodecapeptide motifs. Structural models using X-ray crystallography havebeen generated for I-Cre I, I-Dmo I, PI-Sce I, PI-Pfu I. structures ofI-Cre I bound to its DNA site have also been elucidated leading to anumber of predictions about specific protein-DNA contacts. Seligman etal (Nucleic Acids Research, 2002, 30, 3870-3879) tests these predictionsby analysing a set of endonuclease mutants and a complementary set ofhoming site mutants. In parallel, Gruen et al (Nucleic Acids Research,2002, 30, e29) developed an in vivo selection system to identify DNAtarget site variants that are stik4 by wild-type homing endonucleases.

Endonucleases are requisite enzymes for today's advanced geneticengineering techniques, notably for cloning and analyzing genes. Homingendonucleases are very interesting as rare-cutter endonucleases becausethey have a very low recognition and cleavage frequency in large genomedue to the size of their recognition site. Therefore, the homingendonucleases are used for molecular biology and for geneticengineering.

More particularly, homologous recombination provides a method forgenetically modifying chromosomal DNA sequences in a precise way. Inaddition to the possibility of introducing small precise mutations inorder to alter the activity of the chromosomal DNA sequences, such amethodology makes it possible to correct the genetic defects in geneswhich cause disease. Unfortunately, current methods for achievinghomologous recombination are inherently inefficient, in that homologousrecombination-mediated gene repair can usually be achieved in only asmall proportion of cells that have taken up the relevant “targeting orcorrecting’ DNA. For example, in cultured mammalian cells, suchrecombinational events usually occur in only one in ten thousandtransfected cells.

It has been shown that induction of double stranded DNA cleavage at aspecific site in chromosomal DMA induces a cellular repair mechanismwhich leads to highly efficient recombinational events at that locus.Therefore, the introduction of the double strand break is accompanied bythe introduction of a targeting segment of DNA homologous to the regionsurrounding the cleavage site, which results in the efficientintroduction of the targeting sequences into the locus (either to repaira genetic lesion or to alter the chromosomal DNA in some specific way).Alternatively the induction of a double stranded break at a site ofinterest is employed to obtain correction of a genetic lesion via a geneconversion event in which the homologous chromosomal DNA sequences froman other copy of the gene donates sequences to the sequences where thedouble stranded break was induced. This latter strategy leads to thecorrection of genetic diseases either in which one copy of a defectivegene causes the disease phenotype (such as occurs in the case ofdominant mutations) or in which mutations occur in both alleles of thegene, but at different locations (as is the case of compoundheterozygous mutations). (See WO 96/14408; WO 00/46386; U.S. Pat. No.5,830,729; Choulika et al., Mol Cell Biol, 1995, 15, 1965-73;Cohen-Tannoudji et al., Mol Cell Biol, 15 1998, 18, 1444.8; Donoho etal, Mol Cell Biol; Rouet et al, Mol Cell Biol, 1994, 14, 5096-106; thedisclosure of which is incorporated herein by reference).

Unfortunately, this method of genome engineering by induction ofhomologous recombination by a double stranded break is limited by theintroduction of a recognition and cleavage site of a naturalmeganuclease at the position where the recombinational event is desired.

Up to today, in a first approach for generating new endonuclease, somechimeric restriction enzymes have been prepared through hybrids betweena zinc finger DNA binding domain and the non-specific DNA-cleavagedomain from the natural restriction enzyme Fok I (Smith et al., 2000,Nucleic Acids Res, 28, 3361-9; Smith et al., 1999, Nucleic Acids Res.,27, 274-281v; Kim et al, 1996, Proc Natl Acad Sci USA, 93, 1156-60; Kim& Chandrasegaran, 1994, Proc Natl Acad Sci USA, 91, 883-7; WO 95/109233;WO/9418313).

Another approach consisted of embedding DNA binding and catalyticactivities within a single structural unit, such as type II restrictionendonuclease. However, efforts to increase the length of recognitionsequence or alter the specificity of these enzymes have resulted in theloss of catalytic activity or overall diminution of specificity due tothe tight interdependence of enzyme structure, substrate recognition andcatalysis (Lanio et al, 2000, Protein Eng., 13, 275-281).

Based on homing endonuclease, Chevalier et al. (2002, Molecular Cell,10, 895-905) have generated an artificial highly specific endonucleaseby fusing domains of homing endonucleases I-Dmo I and I-Cre I. Theresulting enzyme binds a long chimeric DNA target site and cleaves itprecisely at a rate equivalent to its natural parents.

However, this experiment leads to one endonuclease with a newspecificity but it is not applicable to find an endonuclease thatrecognizes and cleaves any desired polynucleotide sequence.

Although these efforts, there is still a strong need of new rare-cuttingendonucleases with new sequence specificity for the recognition andcleavage.

The present invention concerns a method for producing a custom-mademeganuclease able to cleave a targeted DNA sequence derived from aninitial meganuclease. This method comprises the steps of preparing alibrary of meganuclease variants and selecting the variants able tocleave the targeted DNA sequence.

In a first embodiment of the method for producing a custom-mademeganuclease, the initial meganuclease is a natural meganuclease.Alternatively, said initial meganuclease is not a natural one.Preferably, said initial meganuclease is a homing endonuclease, morepreferably a LAGLIDADG homing endonuclease. In a more preferredembodiment, said LAGLIDADG homing endonuclease is I-Cre I.

In a second embodiment of the method for producing a custom-mademeganuclease, the library of meganuclease variants is generated bytargeted mutagenesis, by random mutagenesis, by DNA shuffling, bydirected mutation or by a combination thereof. Preferably, said libraryis generated by targeted mutagenesis. Said targeted mutagenesis isperformed in meganuclease segments interacting with the DNA target, andmore preferably introduced at the positions of the interacting aminoacids. Optionally, the amino acids present at the variable positionscomprise or are selected from the group consisting of D, E, H, K, N, Q,R, S, T, Y.

In a particular embodiment of the present invention, a library of I-CreI variants is prepared by introducing amino acid diversity in positionsselected from the group consisting of: Q26, K28, N30, S32, Y33, Q38,Q44, R68, R70 and T140. Preferably, a library of I-Cre I variants isprepared by introducing diversity in positions: a) Q26, K28, N30, Y33,Q38, Q44, R68, R70, T140; b) Q26, K28, N30, Y33, Q38, Q44, R68, R70; c)Q26, K28, N30, Y33, Q44, R68, R70; or d) Q26, K28, Y33, Q38, Q44, R68,R70. More preferably, a library of I-Cre I variants is prepared byintroducing diversity in positions Q26, K28, N30, Y33, Q38, Q44, R68,and R70.

In a third embodiment of the method for producing a custom-mademeganuclease, said selection of the variants able to cleave the targetedDNA sequence or a part thereof comprises the following steps:

a) a selection step for the binding ability, a screening step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity;

b) a selection step for the binding ability, a screening step for thebinding ability, and a screening step for the cleavage activity;

c) a selection step for the binding ability, a selection for thecleavage activity, and a screening step for the cleavage activity; or,

d) a screening step for the binding apathy and a screening step for thecleavage.

Preferably, said selection of the variants able to cleave the targetedDNA sequence or a part thereof comprises a selection step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity, a screening assay for the bindingability after a selection step based on the binding capacity can be donein order to estimate the enrichment of the library for meganucleasevariants presenting a binding capacity.

Preferably, the selection and the screening based on the binding abilityuse the phage display technology. Preferably, the selection based on thecleavage activity uses a test in which the cleavage leads to either theactivation of a positive selection marker or the inactivation of anegative selection marker. Preferably, the screening based on thecleavage activity uses a test in which the cleavage leads to a) theactivation of a positive selection marker or a reporter gene; or b) theinactivation of a negative selection marker or a reporter gene.

Therefore, one object of the present invention is custom-mademeganuclease produced by the above-mentioned method, a polynucleotideencoding said custom made meganuclease and any use thereof. Furthermore,the invention concerns a cell, an animal or a plant comprising saidcustom-made meganuclease or a polynucleotide encoding said custom-mademeganuclease.

The invention concerns the use of a custom-made meganuclease formolecular biology, for in vivo or in vitro genetic engineering for invivo or in vitro genome engineering for antiviral therapy, for genometherapy or for gene therapy.

More particularly, the invention concerns the use of a custom-mademeganuclease for introducing a double-stranded break in a site ofinterest comprising the recognition and cleavage site of saidmeganuclease, thereby inducing a DNA recombination event, preferably ahomologous recombination event, a DNA loss or cell death.

In a first embodiment of a method of genetic engineering, a custom-mademeganuclease introduces a double-stranded break in a site of interestlocated on a vector and comprising the recognition and cleavage site ofsaid meganuclease, thereby inducing a homologous recombination withanother vector presenting homology with the sequence surrounding thecleavage site.

In a second embodiment of a method of genome engineering, the methodcomprises the following steps: 1) introducing a double-stranded break atthe genomic locus comprising at least one recognition and cleavage siteof a custom-made meganuclease according to the present invention; 2)providing a targeting DNA construct comprising the sequence to beintroduced flanked by sequences sharing homologies to the targetedlocus.

In a third embodiment of a method of genome engineering, the methodcomprises the following steps: 1) introducing a double-stranded break atthe genomic locus comprising at least one recognition and cleavage siteof a custom-made meganuclease according to the present invention; 2)maintaining under conditions appropriate for homologous recombinationwith chromosomal DNA sharing homologies to regions surrounding thecleavage site.

These methods of genetic and genome engineering could be used forrepairing a specific sequence, modifying a specific sequence, forattenuating or activating an endogenous gene of interest, forintroducing a mutation into a site of interest, for introducing anexogenous gene, for inactivating or deleting an endogenous gene or apart thereof, for translocating a chromosomal arm, or for killing thecell. The invention relates to the resulting cells and their uses.

Therefore, the invention concerns the use of at least one custom mademeganuclease according to the present invention to repair a specificsequence, to restore a functional gene in place of a mutated one, tomodify a specific sequence, to attenuate or activate an endogenous geneof interest, to introduce a mutation into a site of interest, tointroduce an exogenous gene or a part thereof, and to inactivate ordelete an endogenous gene or part thereof, to translocate a chromosomalarm, or to leave the DNA unrepaired and degraded, by exposing cells,animals, or plants to said meganuclease. Optionally, said cells,animals, or plants are further exposed to a targeting DNA constructcomprising the sequence to be introduced flanked by sequences sharinghomologies to the targeted locus.

The invention also concerns a composition comprising at least onecustom-made meganuclease according to the present invention. Saidcomposition is used for repairing a specific sequence, modifying aspecific sequence for attenuating or activating an endogenous gene ofinterest, for introducing a mutation into a site of interest, forintroducing an exogenous gene or a part thereof, for inactivating ordeleting an endogenous gene or a part thereof, to translocate achromosomal arm, or to leave the DNA unrepaired and by exposing cells,animals, or plants to said meganuclease. Optionally, said compositioncan further comprise a targeting DNA construct comprising the sequenceto be introduced flanked by sequences sharing homologies to the targetedlocus.

The invention also relates to a method for treating or prophylaxis of agenetic disease in an individual in need thereof comprising (a) inducingin cells of the individual a double stranded cleavage at a site ofinterest comprising at least one recognition and cleavage site of acustom-made meganuclease according to the present invention, andintroducing into the individual a targeting DNA, wherein said targetingDNA comprises (1) DNA sharing homologies to the region surrounding thecleavage site and (2) DNA which will be used to repair the site ofinterest in the chromosomal DNA.

In a second embodiment, the method for treating or prophylaxis of agenetic disease in an individual in need thereof comprises inducing incells of the individual a double stranded break at a site of interestcomprising at least one recognition and cleavage site of a custom-mademeganuclease according to the present invention under conditionsappropriate for the DNA homologous to the region surrounding the site ofcleavage to be used in order to repair the site of interest.

The present invention further relates to the resulting cells and theiruses, such as for treatment or prophylaxis of a disease or disorder inan individual.

The present invention concerns the use of at least one custom-mademeganuclease according to the present invention to prevent, ameliorateor cure a genetic disease by exposing cells, animals or patients to saidmeganuclease. Optionally, said cells, animals or patients are alsoexposed to a targeting DNA construct comprising the sequence whichrepairs the site of interest flanked by sequences sharing homologies tothe targeted locus.

The invention concerns a composition comprising at least one custom-mademeganuclease according to the present invention. Preferably saidcomposition is used for preventing, ameliorating or curing a geneticdisease by exposing cells, plants, animals or patients to saidcomposition. Optionally, said composition can further comprise thetargeting DNA construct comprising the sequence which repairs the siteof interest flanked by sequences sharing homologies to the targetedlocus.

The custom meganucleases according to the present invention can also beused as therapeutics in the treatment of diseases caused by infectiousagents that present a DNA intermediate. It is therefore an object of thepresent invention to use at least one custom-made meganuclease accordingto the present invention to prevent, ameliorate or cure infection by aninfectious agent by exposing said infectious agent and/or infectedcells, animals, plants or patients to said meganuclease, the DNA targetsequence of which being present in genome of said infectious agent.Preferably, said infectious agent is a virus.

Another object of the present invention is to use at least onemeganuclease according to the present invention for inactivating ordeleting an infectious agent in biologically derived products andproducts intended for biological uses by treating the products with saidmeganucleases. Preferably, said infectious agent is a virus.

A further object of the invention is a composition comprising at leastone custom-made meganuclease according to the present invention forpreventing, ameliorating or curing an infection by an infectious agentby exposing the infectious agent or the infected cells, animals orpatients to said composition. Preferably, said infectious agent is avirus.

An additional object of the invention is to provide compositionscomprising at least one meganuclease according to the present inventionfor inhibiting propagation of an infectious agent, inactivating ordeleting an infectious agent in biologically derived products orproducts intended for biological use, or for disinfecting an object.Preferably, said infectious agent is a virus.

The invention also relates to a method for treating or prophylaxis of aninfection by an infectious agent in an individual in need thereofcomprising (a) introducing into individual's cells of the individual atleast one custom-mademeganuclease presenting a recognition and cleavagesite in the infectious agent sequence, and (b) inducing a double-strandbreak at said recognition and cleavage site, thereby leading to arecombination event resulting in inactivation or deletion of theinfectious agent.

DEFINITIONS

In the present application, by “meganuclease” is intended adouble-stranded endonuclease having a polynucleotide recognition site of14-40 bp. Said meganuclease is either monomeric or dimeric. Therefore,the meganuclease are also called rare-cutting or very rare cuttingendonuclease. The homing endonucleases are one type of meganucleases.

By “custom-made meganuclease” is intended a meganuclease derived from aninitial meganuclease presenting a recognition and cleavage sitedifferent from the site of the initial one. By “different” is intendedthat the custom-made meganuclease cleaves the site with an efficacity atleast 10 fold more than the natural meganuclease, preferably at least 50fold, more preferably at least 100 fold. The initial meganuclease can bea natural meganuclease or a modified one. By “natural” refers to thefact that an object can be found in nature. For example, a meganucleasethat is present in an organism, that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory is natural.

“Identity” refers to sequence identity between two nucleic acidmolecules or polypeptides. Identity can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base, then the molecules are identical at that position. A degreeof similarity or identity between nucleic acid or amino acid sequencesis a function of the number of identical or matching nucleotides atpositions shared by the nucleic acid sequences. Various alignmentalgorithms and/or programs may be used to calculate the identity betweentwo sequences, including FASTA, or BLAST which are available as a partof the GCG sequence analysis package (University of Wisconsin, Madison,Wis.), and can be used with, e.g., default settings.

By “homologous” is intended a sequence with enough identity to anotherone to lead to a homologous recombination between sequences, moreparticularly having at least 95% identity, preferably 97%, and morepreferably 99%.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. A vector according to the present invention comprises, but isnot limited to, a YAC (yeast artificial chromosome), a BAC (bacterialartificial), a baculovirus vector, a phage, a phagemid, a cosmid, aviral vector, a plasmid, a RNA vector or a linear or circular DNA or RNAmolecule which may consist of chromosomal, non-chromosomal,semi-synthetic or synthetic DNA. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double-stranded DNA loopswhich, in their vector form are not bound to the chromosome. Largenumbers of suitable vectors are known to those of skill in the art andcommercially available, such as the following bacterial vectors: pQE70,pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK,pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress),pET (Novagen).

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.,adenoassociated viruses), coronavirus, negative strand RNA viruses suchas ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabiesand vesicular stomatitis virus), paramyxovirus (e.g., measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D-type viruses, HTLV-BLV group, lenti-virus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, in Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Otherexamples include murine leukemia viruses, murine sarcoma viruses, mousemammary tumor virus, bovine leukemia virus, feline leukemia virus,feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkeyvirus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcomavirus and lentiviruses. Other examples of vectors are described, forexample, in McVey et al., U.S. Pat. No. 5,801,030, the teachings ofwhich are incorporated herein by reference.

Vectors can comprise selectable markers (for example, neomycinphosphotransferase, histidinol dehydrogenase, dihydrofolate reductase,hygromycin phosphotransferase, herpes simplex virus thymidine kinase,adenosine deaminase, glutamine synthetase, and hypoxanthine-guaninephosphoribosyl transferase for eukaryotic cell culture; TRP 1 for S.cerevisiae; tetracycline, rifampicin or ampicillin resistance in E.coli; etc. . . . ). However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

The phrases “site of interest”, “target site” and “specific site”, asused herein, refer to a distinct DNA location, preferably a chromosomallocation, at which a double stranded break (cleavage) is to be inducedby the meganuclease.

As used herein, the term “individual” includes mammals, as well as othervertebrates (e.g., birds, fish and reptiles). The terms “mammal” and“mammalian”, as used herein, refer to any vertebrate animal, includingmonotremes, marsupials and placental, that suckle their young and eithergive birth to living young (eutharian or placental mammals) or areegg-laying (metatharian or nonplacental mammals). Examples of mammalianspecies include humans and other primates (e.g., monkeys, chimpanzees),rodents (e.g., rats, mice, guinea pigs) and ruminants (e.g., cows, pigs,horses).

By “genetic disease” is intended any disease, partially or completely,directly or indirectly, due to an abnormality in one or several genes.Said abnormality can be a mutation, an insertion or a deletion. Saidmutation can be a punctual mutation. Said abnormality can affect thecoding sequence of the gene or its regulatory sequence. Said abnormalitycan affect the structure of the genomic sequence or the structure orstability of the encoded mRNA. Said genetic disease can be recessive ordominant. Such genetic disease could be, but are not limited to, cysticfibrosis, Huntington's chorea, familial hyperchoiesterolemia (LDLreceptor defect), hepatoblastoma, Wilson's disease, congenital hepaticporphyrias, inherited disorders of hepatic metabolism, Lesch Nyhansyndrome, sickle cell anemia, thalassaemias, xeroderma pigmentosum,Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom'ssyndrome, retinoblastoma, Duchenne's muscular dystrophy, and Tay-Sachsdisease.

Generation of Meganuclease Variants

The present invention concerns a method to produce a custom-mademeganuclease specific to a targeted DNA sequence derived from an initialmeganuclease by the introduction of diversity. Optionally, said initialmeganuclease is a natural meganuclease. This method comprises the stepsof preparing a library of meganuclease variants and isolating, byselection and/or screening, the variants able to bind and/or cleave thetargeted DNA sequence or a part thereof.

The diversity could be introduced in the meganuclease by any methodavailable for the man skilled in the art. Preferably, the diversity isintroduced by targeted mutagenesis (i.e. cassette mutagenesis,oligonucleotide directed codon mutagenesis, targeted randommutagenesis), by random mutagenesis (i.e. mutator strains, Neurosporacrassa system (U.S. Pat. No. 6,232,112; WO01/70946, error-prone PCR), byDNA shuffling, by directed mutation or a combination of thesetechnologies (See Current Protocols in Molecular Biology, Chapter 8“Mutagenesis in cloned DNA”, Eds Ausubel et al., John Wiley and Sons).The meganuclease variants are preferably prepared by the targetedmutagenesis of the initial meganuclease. The diversity is introduced atpositions of the residues contacting or interacting directly orindirectly with the DNA target. The diversity is preferably introducedin regions interacting with the DNA target, and more preferablyintroduced at the positions of the interacting amino acids. In librariesgenerated by targeted mutagenesis, the 20 amino acids can be introducedat the chosen variable positions. Preferably, the amino acids present atthe variable positions are the amino acids well-known to be generallyinvolved in protein-DNA interaction. More particularly, these aminoacids are generally the hydrophilic amino acids. More preferably, theamino acids present at the variable positions comprise D, E, H, K, N, Q,R, S, T, Y. Optionally, the amino acids present at the variablepositions are selected from the group consisting of D, E, H, K, N, Q, R,S, T, Y. Synthetic or modified amino acids are also contemplated in thepresent invention.

One preferred way to generate a directed library is the use ofdegenerated codons at the positions where diversity has to beintroduced. Several types of degenerated codons could be used. Adegenerated codon N N K ([ATCG] [ATCG] [TG]) leads to 32 differentcodons encoding the 20 amino acids and one stop. A degenerated codon N VK ([ATCG] [ACG] [TG]) leads to 24 different codons encoding the 15 aminoacids and one stop. A degenerated codon V V K ([ACG] [ACG] [TG]) leadsto 18 different codons encoding the 12 amino acids (A, D, E, G, H, K, N,P, Q, R, S, T) and no stop. A degenerated codon R V K ([AG] [ACG] [TG])leads to 12 different codons encoding the 9 amino acids (A, D, E, G, K,N, R, S, T). Preferably, a degenerated codon V V K ([ACG] [ACG] [TG])leading to 18 different codons encoding the 12 amino acids (A, D, E, G,H, K, N, P, Q, R, S, T) is used for generating the library. Indeed, theV V K degenerated codon does not contain any stop codon and comprisesall the hydrophilic amino acids.

If a directed library is generated, knowledge on amino acidsinter-acting with the DNA target is useful. This knowledge could beprovided, for example, by X-ray cristallography, Alanine scanning, orcross-linking experiments. The amino acids interacting with the DNAtarget can also be deduced by sequence alignment with a homologousprotein.

The custom-made meganuclease is derived from any initial meganuclease.By initial meganuclease is intended a natural one or a modified one.Said modified one can be derived from natural ones by the hybridgeneration or by a modification of physico-chemical properties of anatural one. Optionally, the initial meganuclease is selected so as itsnatural recognition and cleavage site is the closest to the targeted DNAsite. Preferably, the initial meganuclease is a homing endonuclease, asspecified, in the here above definitions. Homing endonucleases fall into4 separated families on the basis of well conserved amino acids motifs,namely the LAGLIDADG family, the GIY-YIG family, the His-Cys box family,and the HNH family (Chevalier et al., 2001, N.A.R, 29, 3757-3774).

The detailed three-dimensional structures of several homingendonucleases are known, namely I-Dmo I, PI-See I, PI-Pfu I, I-Cre I,I-Ppo I, and a hybrid homing endonuclease I-Dmo I/I-Cre I called E-Dre I(Chevalier et al., 2001, Nat Struct Biol, 8, 312-316; Duan et al., 1997,Cell, 89, 555-564; Heath et al., 1997, Nat Struct Biol, 4, 468-476; Huet al., 2000, J Biol Chem, 275, 2705-2712; Ichiyanagi et al., 2000, JMol Biol, 300, 889-901; Jurica et al., 1998, Mol Cell, 2, 469-476;Poland et al., 2000, J Biol Chem, 275, 16408-16413; Silva et al., 1999,J Mol Biol, 286, 1123-1136; Chevalier et al., 2002, Molecular Cell, 10,895-905).

The LAGLIDADG family is the largest family of proteins clustered bytheir most general conserved sequence motif: one or two copies of atwelve-residue sequence: the di-dodecapeptide, also called LAGLIDADGmotif. Homing endonucleases with one dodecapeptide (D) are around 20 kDain molecular mass and act as homodimer. Those with two copies (DD) rangefrom 25 kDa (230 AA) to 50 kDa (HO, 545 AA) with 70 to 150 residuesbetween each motif and act as monomer. Cleavage is inside therecognition site, leaving 4 nt staggered cut with 3′OH overhangs. I-CeuI, and I-Cre I illustrate the homodimeric homing endonucleases with oneDodecapeptide motif (mono-dodecapeptide). I-Dmo I, I-Sce I, PI-Pfu I andPI-Sce I illustrate monomeric homing endonucleases with twoDodecapeptide motifs.

The initial LAGLIDADG homing endonuclease can be selected from the groupconsisting of: I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Sce I,PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-CtrI, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I,PI-Mfl I, PI-Mga I, PI-Mgo I, PI Min I, PI-Mka I, PI-Mle I, PI-Mma I,PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I,PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I,PI-Thy I, PI-Tko I, and PI-Tsp I; preferably, I-Sce I, I-Chu I, I-Dmo I,I-Cre I, I-Csm I, PI-Sce I, PI-Pfu I, PI-Tli I, PI-Mtu I, and I-Ceu I;more preferably, I-Dmo I, I-Cre I, PI-Sce I, and PI-Pfu I; still morepreferably I-Cre I.

The four structures of LAGLIDADG homing endonucleases, namely those ofI-Dmo I, PI-Sce I, PI-Pfu I, and I-Cre I, reveal the functionalsignificance of the LAGIDADG motif, and the nature of the DNA-bindinginterface. The core a α β β α β β α a fold of the homodimer homingendonuclease is repeated twice in the monomer homing endonuclease andconfers upon the monomer a pseudo-dimeric structure. The first α-helixof each domain or subunit contains the defining LAGLIDADG motif. The twoLAGLIDADG helices of each protein form a tightly packed dimer or domaininterface. The DNA binding interface is formed by the four β-strands ofeach domain or subunit that fold into an antiparallel β sheet. A minimalDNA binding moiety could be defined in the LAGLIDADG homingendonucleases as a β-hairpin (2 β-strands connected by a loop or turn),two such β-hairpins being connected into the 4-stranded β-sheet.

Each domain or subunit interacts with a half recognition site. The<<external>> quarter recognition site can be defined by its interactionwith only one of the 2 β-hairpins of each domain or subunit.

Therefore, meganuclease variants derived from LAGLIDADG homingendonuclease can be fragmented in several directed libraries. Thisfragmented approach for the evolution of an initial meganuclease allowsthe introduction of a greater diversity (more amino acids at a positionand/or more diversificated positions). In each library, the diversity isintroduced only in the region involved in the interaction with a half ora quarter recognition site, the targeted DNA being modified only for thepart interacting with the region comprising the introduced diversity.More particularly, if a new half site is searched for, then thediversity is preferably introduced in the 4-stranded β-sheet of onedomain or subunit, more preferably at the positions of the DNAinteracting amino acids in this structure. If a new quarter site issearched for, then the diversity is introduced in the correspondingβ-hairpin, more preferably at the positions of the DNA interacting aminoacids of this structure.

Preferably, a set of libraries covers the entire targeted DNA site.Hence, if the libraries comprise diversity only in the regioninteracting with a half site, at least two libraries, preferably two,are necessary. However, if the initial meganuclease is a dimer, onelibrary is enough with a half-site approach. If the libraries comprisediversity only in the region interacting with a quarter site, at leastfour libraries, preferably four, are necessary. If the initialmeganuclease is a dimer, two libraries can be enough with a quarter siteapproach.

After the selection or screening of the primary libraries, the selectedelements from the primary libraries are fused or combined in asubsequent library for a new cycle of selection. For example, twolibraries can be fused by shuffling. A new cycle of selection could bethen done on the whole targeted DNA site. Optionally, the new cycle ofselection can be done on a half targeted DNA site if the first librariesare based on a quarter site. Subsequently, the results of the selectionand/or screening of the half site are combined to give a final librarywhich can be screened for the whole targeted DNA site.

Alternatively, the best elements from each libraries are joined togetherin order to obtain a meganuclease able to bind and cleave the targetedDNA site.

In another approach, a library with diversity located only in the regioninvolved in the interaction with a half or a quarter recognition site isprepared. Then, after selection or screening of this library, theselected elements from the library are modified such as to introducediversity in another region involved in the inter action withrecognition site, leading to a subsequent library. Libraries aregenerated until the complete targeted DNA site is bound and cleaved bythe selected meganuclease.

More specifically, for the dimeric homing endonuclease (such as I Cre Iand I-Ceu I), a library can be generated by introducing diversity onlyin the region interacting with a half-site, a half site corresponding toone monomer of the initial homing endonuclease. This library can be usedfor selection and/or screening on each half sites of the target DNAsequence. When positive elements from the library have been selected foreach half site, a variant for the first half site and a variant for theother half site are brought together for binding and cleaving the wholetarget DNA sequence. Alternatively, the positive variants can beintroduced in a single chain meganuclease structure. As described inExample 1, a single chain meganuclease is an enzyme in which the twomonomers of the initial dimeric homing endonuclease are covalently boundby a linker.

If an approach by a quarter site is chosen from an initial dimer homingendonuclease, at least two libraries are generated by introducingdiversity only in the region involved in the interaction with eachquarter recognition sites. After the selection or screening of theprimary libraries, the selected variants from the primary libraries arefused in a subsequent library for a new cycle of selection on the halfsite. Alternatively, the best elements from each libraries are joinedtogether to obtain a monomer able to bind the half site. Otherwise, alibrary with diversity only in the region involved in the interactionwith a quarter recognition site is prepared. Then, after selection orscreening of this library, the selected elements from the library aremodified such as to introduce diversity in the region involved in theinteraction with the other quarter site, leading to a subsequentlibrary. The selection and/or screening of this second library lead tothe variants monomer able to bind the half site. When positive elementsfrom the library have been selected for each half sites, a variant forthe first half site and a variant for the other half site are broughttogether for binding and cleaving the target DNA sequence.Alternatively, the positive variants can be introduced in a single chainmeganuclease structure.

In a preferred embodiment, the present invention concerns a method toprepare a custom-made meganuclease which recognizes and cleaves adesired polynucleotide target is derived from the directed evolution ofthe homing endonuclease I-Cre I. As the homing endonuclease is ahomodimer, the approach in this case is based either on the halfrecognition site or on the quarter site.

The directed evolution is based on a library of I-Cre I variants. TheseI-Cre I variants present a diversity of amino acids at several positionspredicted to interact with the polynucleotide target.

The X-ray structure of I-Cre endonuclease with its DNA target predictedthat the following positions are involved: Q26, K28, N30, S32, Y33, Q38,Q44, R68, R70 and T140. Seligman et al (supra) showed that the positionsS32 and T140 appear to be relatively unimportant for DNA recognition.

In one embodiment of the present invention, a set of I-Cre I variants isprepared by introducing amino acid diversity in positions selected fromthe group consisting of: Q26, K28, N30, S32, Y33, Q38, Q44, R68, R70 andT140. In a preferred embodiment, a set of I-Cre I variants is preparedby introducing diversity in positions: a) Q26, K28, N30, Y33, Q38, Q44,R68, R70, T140; b) Q26, K28, N30, Y33, Q38, Q44, R68, R70; c) Q26, K28,N30, Y33, Q44, R68, R70; or d) Q26, K28, Y33, Q38, Q44, R68, R70.Preferably, a set of I-Cre I variants is prepared by introducingdiversity in positions Q26, K28, N30, Y33, Q38, Q44, R68, and R70.

Optionally, the residue D75 of I-Cre I could be mutated in an unchargedamino acid such as N. Indeed, this amino acid has an interaction with 2residues which are preferably modified in the library. As this charge ispresent in the core of the structure, it could be preferable to abolishthis charge.

If the evolution approach of the homing endonuclease I-Cre I is based onthe quarter recognition site, replacing the DNA binding residuespresented by a β-hairpin (within the 4-stranded b-sheet) is a practicalsolution. As those residues are part of an element with limited length(i.e. less than 25 residue), they can be mutated together at once, forexample by cassette replacement. Visual inspection of structure 1g9y(I-Cre I with its target double-stranded DNA) indicates that the firstβ-hairpin is a unique or major contributor to the recognition of thelast six bases of the target (i.e. either bases −12 to −7 or bases +7 to+12). Thus replacing the sequence from residue S22 to residue Q44, morepreferably from residue 124 to residue T42, should be sufficient tospecify new interaction specificity for the last six bases of the targetsite. More preferably, the residues interacting directly with DNA shouldbe modified: I24, Q26, K28, N30, S32, Y33, Q38, S40 and T42.Alternatively (or in addition), the turn at the middle of the β-hairpin,which interacts with the very end of the 24 bp-long DNA target, may bereplaced by a short and flexible loop that would be tolerant to DNAbases substitution. For example, residues 30 to 36 could be replaced by2, 3, 4, 5 or 6 glycine residues. This strategy is worth testing withall meganucleases presenting a comparable 3D structure. The secondhairpin could be replaced similarly as a single unit (from residue Y66to I77). However, while this hairpin interacts predominantly with theinternal quarter site (bases −6 to −1 or +1 to +6), other residues (i.e.S22, Q44 and T46) separated from the hairpin may play a role indirecting the specificity of interaction. Thus, a library could becreated by replacing residues Y66, R68, R70, V73, D75 and I77. Inparallel, S22, Q44 and T46 may either be left untouched, replaced bysmall polar amino acids (G, S or T; more preferably S or T), orrandomized to contribute to the library. Mutants selected from separatelibrary (the first wherein randomized residues are I24, Q26, K28, N30,S32, Y33, Q38, S40 and T42 and the second wherein randomized residuesare Y66, R68, R70, V73, D75 and 177) can be combined together bystandard DNA shuffling methods based on recombination at homologous DNAregions (i.e. the DNA coding for the region between residue 43 andresidue 65 is strictly conserved). However, if the second libraryincludes mutations of residues S22, Q44 and T46, recombination becomesimpractical, and more classical DNA/protein engineering is required.

If the evolution approach of the homing endonuclease I-Cre I is based onthe quarter recognition site, a library of I-Cre I variants is preparedby introducing diversity in positions selected from the group consistingof: a) I24, Q26, K28, N30, S32, Y33, Q38, S40 and T42; or b) Y66, R68,R70, V73, D75, and I77. In the alternative b), the diversity could bealso introduced in positions selected from the group consisting of: S22,Q44, and T46.

Alternatively, a custom-made meganuclease which recognizes and cleaves adesired polynucleotide target could be prepared by the directedevolution of single chain I-Cre I endonuclease. A set of single-chainI-Cre I variants is prepared by introducing amino acid diversity inpositions selected from the group consisting of: Q26, K28, N30, S32,Y33, Q38, Q44, R68, R70, Q123, K125, N127, S129, Y130, Q135, Q141, R165,R167.

Selection and Screening

Two properties of the meganuclease can be used for the steps ofselection and/or screening, namely the capacity to bind the targeted DNAsequence and the ability to cleave it.

The meganuclease variants can be selected and screened, or onlyscreened. The selection and/or screening can be done directly for theability of the meganuclease to cleave the targeted DNA sequence.Alternatively, the selection and/or screening can be done for thebinding capacity on the targeted DNA sequence, and then for ability ofthe meganuclease to cleave it. Preferably, the method to prepare acustom-made meganuclease comprises or consists of the following steps:

a) a selection step for the binding ability, a screening step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity;

b) a selection step for the binding ability, a screening step for thebinding ability, and a screening step for the cleavage activity;

c) a selection step for the binding ability, a selection for thecleavage activity, and a screening step for the cleavage activity;

d) a screening step for the binding ability and a screening step for thecleavage activity;

e) a selection step for and a screening step for the cleavage activity;or,

f) a screening step for the cleavage activity.

More preferably, the method to prepare a custom-made meganucleasecomprises or consists of the following steps: a selection step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity. A screening assay for the bindingability after a selection step based on the binding capacity can be donein order to estimate the enrichment of the library for meganucleasevariants presenting a binding capacity.

The selection and screening assays are performed on the DNA region inwhich a double-stranded cleavage has to be introduced or a fragmentthereof. Preferably, the targeted sequences comprise at least 15nucleotides, preferably 18 to 40, more preferably 18 to 30 nucleotides.In case of dimeric meganuclease, the targeted DNA polynucleotide can bereduced to at least 8 nucleotides for binding only. Preferably, thetargeted DNA polynucleotide length is less than 10 kb, preferably lessthan 3 kb, more preferably less than I. kb. For the DNA binding assay,the targeted DNA polynucleotide length is preferably less than 500 bp,more preferably less than 200 bp.

Any targeted sequence can be used to generate a custom-made meganucleaseable to cleave it according. Optionally, the targeted sequence is chosensuch as to present the most identity with the original recognition andcleavage site of the initial meganuclease.

Therefore, the DNA region in which a double-stranded break has to beintroduced is analyzed to choose at least 1, 2, 3 or 5 sequences of atleast 15 nucleotides length, preferably 18 to 40 nucleotides, morepreferably 18 to 30 nucleotides, having at least 25% identity,preferably 50% identity and more preferably 75% identity with theoriginal recognition and cleavage site of the initial meganuclease.

The targeted DNA sequence is adapted to the type of meganucleasevariants library. If the Library is based on a half site approach, thetargeted DNA sequence used for the selection/screening comprises onehalf original site and one half site of the desired DNA sequence. If thelibrary is based on a quarter site approach, the targeted DNA sequenceused for the selection/screening comprises three quarters of theoriginal site and one quarter site of the desired DNA sequence.

The meganuclease variants resulting from the selection and/or screeningsteps could optionally be an input for another cycle of diversityintroduction.

The positive meganuclease variants selected by the selection and/orscreening steps are validated by in vitro and/or ex vivo cleavage assay.

Selection and/or Screening Based on Binding Property of Meganuclease

The selection and screening of meganuclease variants based on thebinding capacity has to be made in conditions that are not compatiblewith the cleavage activity. For example, most of homing endonucleasesneed manganese or magnesium for their cleavage activity. Therefore, thebinding assays on this type of homing endonuclease variants are donewithout manganese or magnesium, preferably replaced by calcium.

Selection Based on Binding Property of Meganuclease

The binding selection assay is based on the enrichment of themeganuclease variants able to bind the targeted DNA polynucleotide.Therefore, the meganuclease variants encoded by the library areincubated with an immobilized targeted DNA polynucleotide so thatmeganuclease variants that bind to the immobilized targeted DNApolynucleotide can be differentially partitioned from those that do notpresent any binding capacity. The meganuclease variants which are boundto the immobilized targeted DNA polynucleotide are then recovered andamplified for a subsequent round of affinity enrichment andamplification. After several rounds of affinity enrichment andamplification, the library members that are thus selected can beisolated. Optionally, the nucleotide sequences encoding the selectedmeganuclease variants are determined, thereby identifying of themeganuclease variants able to bind the targeted DNA sequence.

The selection of meganuclease variants requires a system linkinggenotype and phenotype such as phage display (W091/17271, W091/18980,and W091/19818 and W093/08278; the disclosures of which are incorporatedherein by reference), ribosome display (Hanes & Plückthun, PNAS, 1997,vol. 94, 4937-4942; He & Taussig, Nucl. Acids Res. (1997) vol. 25, p5132-5143) and mRNA-protein fusion (WO00147775; U.S. Pat. No. 5,843,701;Tabuchi et al FEBS Letters 508 (2001) 309-312; the disclosures of whichare incorporated herein by reference).

Phage display involves the presentation of a meganuclease variant on thesurface of a filamentous bacteriophage, typically as a fusion with abacteriophage coat protein. The library of meganuclease variants isintroduced into a phage chromosome or phagemid so as to obtain a proteinfusion with a bacteriophage coat protein, preferably with the pIIIprotein. If the initial meganuclease is a homodimer, the monomervariants of the meganuclease are introduced so as to be displayed andthe constant monomer can be introduced so as to be produced in theperiplasm. The bacteriophage library can be incubated with animmobilized targeted DNA sequence so that elements able to bind the DNAare selected.

mRNA-protein fusion system opens the possibility to select among 10¹³different meganuclease variants. This system consists in the creation ofa link between the mRNA and the encoded protein via a puromycin at the3′ end of the mRNA which leads to a covalent mRNA-protein fusion at theend of the translation. Hence, a double-stranded DNA library comprisingthe coding sequence for the meganuclease variants is used to regeneratemRNA templates for translation that contain 3′ puromycin. ThemRNA-puromycin conjugates are translated in vitro to generate themRNA-meganuclease fusions. After cDNA synthesis, the fusions are testedfor the ability to bind the immobilized targeted DNA polynucleotide. APCR is then used to generate double-stranded DNA enriched inmeganuclease variants presenting the binding capacity. If the initialmeganuclease is a homodimer, the constant monomer can be introducedeither as DNA or mRNA encoding this monomer or as a monomer protein. Inthis case, an approach with the single chain meganuclease will bepreferably used.

Ribosome display involves a double-stranded DNA library comprising thecoding sequence for the meganuclease variants that is used to generatemRNA templates for translation. After a brief incubation, translation ishalted by addition of Mg²⁺ and incubation at a low temperature oraddition of translation inhibitor. The ribosome complexes are thentested for the ability to bind immobilized targeted DNA polynucleotide.The selected mRNA is used to construct cDNA and a PCR generatesdouble-stranded DNA enriched in meganuclease variants presenting thebinding capacity. If the initial meganuclease is a homodimer, theconstant monomer is introduced either as DNA or mRNA encoding thismonomer or as a monomer protein. In this case, an approach with thesingle chain meganuclease will be preferably used.

The targeted DNA sequence can be immobilized on a solid support. Saidsolid support could be a column, paramagnetic beads or a well of amicroplate. For example, the polynucleotides comprising the targeted DNAsequence present a ligand (such as a biotin) at one end, said ligandallowing the immobilization on a solid support bearing the target of theligand (for example, streptavidin if biotin is used).

The selection of the meganuclease variants may usually be monitored by ascreening assay based on the binding or cleavage capacity of thesemeganucleases. However, the selected meganuclease variants can be alsodirectly introduced in a selection step based on the cleavage capacity.

Screening Based on Binding Property of Meganuclease

In order to perform the screening assay, the selected meganucleasevariants need to be cloned. If the selection was done with the phagedisplay system, the clone encoding each meganuclease variants can beeasily isolated. If the selection was done by mRNA-protein fusion orribosome display, the selected meganuclease variants have to besubcloned in expression vector.

The screening assays are preferably performed in microplates (96, 384 or1536 wells) in which the targeted DNA polynucleotides are immobilized.After expression of the meganuclease variants, these variants areincubated with the immobilized targeted DNA polynucleotides. Themeganuclease variants expression can be performed either in vivo or invitro, preferably by in vitro expression system. Preferably, themeganuclease variants are purified prior to the incubation with thetargeted polynucleotide. The retained meganuclease variants are thendetected. The detection could be done by several means well known by theman skilled in the art. For example, if phages are used, the detectioncan be done with antibodies against phages (ELISA). Otherwise, theexpression could be done in presence of S35 amino acids in order toobtain radioactive meganucleases. Thus, the binding is estimated by aradio-activity measurement. The invention also considers the othersmeans of detection of DNA binding by meganuclease available to the manskilled in the art.

Optionally, the nucleotide sequences encoding the positively screenedmeganuclease variants are determined, thereby identifying of themeganuclease variants able to bind the targeted DNA sequence.

The positively screened meganuclease variants have to be tested fortheir cleavage capacity. Therefore, said meganuclease variants areincorporated in a cleavage selection and/or screening experiment,preferably an in vivo cleavage screening assay. Optionally, saidmeganuclease variants can be tested by an in vitro cleavage assay.

The screening assay can also be used only for estimate the enrichment inmeganuclease variants presenting the binding capacity. This estimationhelps to decide if a new round of selection based on the bindingcapacity is necessary or if the selected library can be submitted to acleavage selection and/or screening, preferably an in vivo cleavageselection and/or screening.

Selection and/or Screening Based on Cleavage Property of Meganuclease

The selection and screening of meganuclease variants based on thecleavage capacity has to be made in conditions compatible with thecleavage activity. The meganuclease variants used in the selectionand/or screening based on cleavage capacity may be either the initiallibrary of meganuclease variants or the meganuclease variants selectedand/or screened for the binding activity.

If necessary, the selected and/or screened meganuclease variants aresubcloned in an appropriate expression vector for the in vitro and invivo cleavage assay. Such subcloning step can be performed in batch orindividually. More particularly, if the initial meganuclease is a dimer,the subcloning step allows the introduction of the selected library(ies)in a single chain meganuclease structure. If two libraries have beenselected and/or screened for two half recognition and cleavage sites,the subcloning step allows to bring together the two selected librariesin a single chain meganuclease structure.

Selection Based on Cleavage Property of Meganuclease

The general principle of an in vivo selection of the meganucleasevariants based on their cleavage capacity is that the double-strandbreak leads to the activation of a positive selection marker or theinactivation of a negative selection marker.

If the selection is based on the inactivation of a negative selectionmarker, the method involves the use of cell containing an expressionvector comprising the coding sequence for a negative selection markerand the targeted DNA sequence for the desired meganuclease and anexpression vector comprising the library of meganuclease variants.Preferably said expression vector is a plasmid. Preferably said targetedDNA sequence is located either near the negative selection gene or inthe negative selection gene, preferably between the promoter driving theexpression of the negative selection and the ORF. The expression of thenegative selection marker has to be conditional in order to keep thecell alive until the meganuclease variants have the opportunity tocleave. Such a conditional expression can be easily done with aconditional promoter. However, there are other conditional systems thatcould be used. The meganuclease variants are introduced in an expressioncassette. The meganuclease encoding sequence can be operably linked toan inducible promoter or to a constitutive promoter. Of course, thepromoter is compatible with the cell used in the assay. If themeganuclease variant has the capacity to cleave the targeted DNA, thenthe negative selection marker is inactivated, either by deleting thewhole negative marker gene or a part thereof (coding sequence orpromoter) or by degrading the vector. A culture in a negative selectioncondition allows the selection of the cell containing the meganucleasevariants able to cleave the targeted DNA sequence.

The vector comprising the negative selection marker is preferablytransfected before the introduction of the vector encoding themeganuclease variants. Optionally, the vector comprising the negativeselection marker can be conserved in the cell in an episomal form.Alternatively, the vector comprising the negative selection marker andthe vector encoding the meganuclease variants can be cotransfected intothe cell. The cell can be prokaryotic or eukaryotic. Preferably, theprokaryotic cell is E. coli. Preferably, the eukaryotic cell is a yeastcell. The negative selection marker is a protein directly or indirectlytoxic for the cell. For example, the negative selection marker can beselected from the group consisting of toxins, translation inhibitors,barnase, and antibiotic for bacteria, URA3 with 5FOA (5-fluoro-oroticacid) medium and LYS2 with a α-AA medium (alpha-adipic acid) for yeast,and thymidine kinase for superior eukaryotic cells. For an example ofnegative marker selection, see Gruen et al., 2002, Nucleic AcidsResearch, 30, e29; the disclosure of which is incorporated herein byreference.

If the selection is based on the activation of a positive selectionmarker, the method involves the use of cell containing an expressionvector comprising an inactive positive selection marker and the targetedDNA sequence for the desired meganuclease and an expression vectorcomprising the library of meganuclease variants. Optionally, theinactive positive selection marker, the targeted DNA sequence and thelibrary of meganuclease variants can be on the same vector (See WO02/44409). Preferably said expression vector is a plasmid. Themeganuclease variants are introduced in an expression cassette. Themeganuclease encoding sequence can be operably linked to an induciblepromoter or to a constitutive promoter. Of course, the promoter iscompatible with the cell used in the assay. For example, the positiveselection marker can be an antibiotic resistance (e.g. tetracycline,rifampicin and ampicillin resistance) or an auxotrophy marker forbacteria, TRP 1, URA3, or an auxotrophy marker for yeast, and neomycineand puromycine for superior eukaryotic cell. Optionally, the positiveselection marker can be an auxotrophy marker compatible with bothbacteria and yeast (e.g. URA3, LYS2, TRP 1, and LEU2). The inactivepositive selection marker gene and the targeted DNA sequence have to bearranged so that the double-strand break leads to a rearrangement of themarker in an active positive marker. Two kinds of repair processes canlead to an active positive selection marker, namely single-strandannealing (SSA) or gene conversion (GC).

The in vivo Single-strand annealing recombination test (SSA) is known bythe man skilled in the art and disclosed for example in Rudin et al.(Genetics 1989, 122, 519-534; Fishman-Lobell & Haber (Science 1992, 258,480.4); Lin et al (Mol. Cell. Biol., 1984, 4, 1020-1034) and Rouet et al(Proc. Natl. Acad. Sci. USA, 1994, 91, 6064-6068); the disclosure ofwhich are incorporated herein by reference.

To test the meganuclease variants, an in vivo assay based on SSA in acell, preferably a bacterial or yeast cell has been developed. Forinstance, the method uses a yeast cell. This organism has the advantagethat it recombines naturally its DNA via homologous recombination with ahigh frequency.

This in vivo test is based on the reparation by SSA of a positiveselection marker induced by double-strand break generated by an activemeganuclease variant. The target consists of a modified positiveselection gene with an internal duplication separated by an interveningsequence comprising the targeted DNA sequence. The internal duplicationshould contain at least 50 bp, preferably at least 200 bp. Theefficiency of the SSA test will be increased by the size of the internalduplication. The intervening sequences are at least the targeted DNAsequence. The intervening sequence can optionally comprise a selectionmarker, this marker allowing checking that the cell has not repaired thepositive selection marker by a spontaneous recombination event. Thepositive selection marker gene is preferably operably linked to aconstitutive promoter relating to the cell used in the assay. Accordingto said assay method, the cell will be selected only if an SSA eventoccurs following the double-strand break introduced by an activemeganuclease variant.

Optionally, each vector can comprise a selectable marker to ensure thepresence of the plasmid in the cell. The presence of this selectablemarker is preferable for the assay performed in yeast cell. For example,for yeast, a first construct comprising the target gene can comprise aLeu2 selectable marker allowing transformed yeast to grow on a syntheticmedium that does not contain any Leucine and a second construct cancomprise the Trp1 selectable marker allowing transformed yeast to growon a synthetic medium that does not contain any tryptophane.

The vector comprising the positive selection marker is preferablytransfected before the introduction of the vector encoding themeganuclease variants. Optionally, the vector comprising the positiveselection marker can be conserved in the cell in an episomal form.Alternatively, the vector comprising the positive selection marker andthe vector encoding the meganuclease variants can be cotransfected intothe cell.

The in vivo selection of the meganuclease variants can also be performedwith a gene conversion assay. For example, the selection vectorcomprises a first modified positive selection gene with a deletion or amutation and an insertion of the targeted DNA sequence for themeganuclease at the place of the deletion. The positive selection genecan also be inactivated by the interruption of the gene by an insertcomprising the targeted DNA sequence. The selection construct furthercomprises the segment of the positive selection marker gene which hasbeen deleted flanked at each side by the positive selection marker genesequences bordering the deletion. The bordering sequences comprise atleast 100 bp of homology with the positive selection marker gene at eachside, preferably at least 300 pb. The double-stand break generated by anactive meganuclease variant in the targeted DNA sequence triggers on agene conversion event resulting in a functional positive selectionmarker gene. This kind of assay is documented in the following articles:Rudin et al (Genetics 1989, 122, 519-534), Fishman-Lobell & Haber(Science 1992, 258, 480 4), Paques & Haber (Mol. Cell. Biol., 1997, 17,6765-6771), the disclosures of which are incorporated herein byreference.

Otherwise, the in vivo selection of the meganuclease variants can beperformed through a recombination assay on chromosomic target. Therecombination can be based on SSA or gene conversion mechanisms. The invivo selection can be based on several SSA targets, preferably at leasttwo SSA targets.

A first example based on SSA is the following. A modified positiveselection gene with an internal duplication separated by an interveningsequence comprising the targeted DNA sequence for the desiredmeganuclease variant is introduced into the chromosome of the cell. Theinternal duplication should contain at least 50 bp, preferably at least200 bp. The efficiency of the SSA test will be increased by the size ofthe internal duplication. The intervening sequence is at least thetargeted DNA sequence. By transfecting the cell with an expressionconstruct allowing the production of a meganuclease variant in the cell,the repair by homologous recombination of the double-strand breakgenerated by an active meganuclease variant will lead to a functionalpositive selection marker gene.

Another example based on gene conversion is the following. A mutatednon-functional positive selection marker gene comprising the targetedDNA sequence for the desired meganuclease variant is introduced into thechromosome of the cell. Said targeted DNA sequence has to be in thevicinity of the mutation, preferably at less than 1 kb from themutation, more preferably at less than 500 bp, 200 bp, or 100 pbsurrounding the mutation. By transfecting the cell with a fragment ofthe functional positive selection marker gene corresponding to themutation area and an expression construct allowing the production of ameganuclease variant in the cell, the repair by homologous recombinationof the double-strand break generated by an active meganuclease variantwill lead to a functional positive selection marker gene. Alternatively,the fragment of the functional positive selection marker allowing therepair can be integrated on the chromosome. This kind of assay isdocumented in the following articles: Rouet et al (Mol. Cell. Biol.,1994, 14, 8096-8106); Choulika et al (Mol. Cell. Biol., 1995, 15,1968-1973); Donoho et al (Mol. Cell. Biol., 1998, 18, 4070-4078); thedisclosures of which are incorporated herein by reference.

The selected clones comprise a meganuclease variant presenting thecapacity to cleave the targeted DNA sequence. It is preferable tovalidate the selection by a screening assay. This screening assay can beperformed in vivo or in vitro, preferably in vivo.

Optionally, the nucleotide sequences encoding the positively screenedmeganuclease variants are determined, thereby identifying themeganuclease variants able to cleave the targeted DNA sequence.

Screening Based on Cleavage Property of Meganuclease

In order to perform the screening assay, the selected meganucleasevariants need to be cloned and the cleavage assay need to be performedindividually for each clone.

The in vivo cleavage assay for the screening is similar to those usedfor the selection step. It can be based on the inactivation of either anegative selection marker or a reporter gene, or on the activation ofeither a positive selection marker or a reporter gene.

By reporter gene is intended any nucleic acid encoding a product easilyassayed, for example β-galactosidase, luciferase, alkaline phosphatase,green fluorescent protein, tyrosinase, DsRed proteins. The reporter geneis preferably operably linked to a constitutive promoter relating to thecell used in the assay (for example CMV promoter).

Cells used for this screening assay can be prokaryotic, preferably E.coli, or eukaryotic, preferably a yeast cell or a mammalian cell. Moreparticularly, it could be interesting to use mammalian cells for avalidation of a positive meganuclease variant by an ex vivo cleavageassay.

In Vitro Cleavage Assay

The recognition and cleavage of the targeted DNA sequence or a partthereof by the meganuclease variants can be assayed by any method knownby the man skilled in the art.

One way to test the activity of the meganuclease variants is to use anin vitro cleavage assay on a polynucleotide substrate comprising thetargeted DNA sequence or a part thereof. Said polynucleotide substratecould be a synthetic target site corresponding to:

-   -   the whole targeted DNA site;    -   a half targeted DNA site and a half original site; or,    -   a quarter targeted DNA site and three quarters original site.

Said polynucleotide substrate can be linear or circular and comprisespreferably only one cleavage site. The assayed meganuclease variant isincubated with the polynucleotide substrate in appropriate conditions.The resulting polynucleotides are analyzed by any known method, forexample by electrophoresis on agarose or by chromatography. If thepolynucleotide substrate is a linearized plasmid, the meganucleaseactivity is detected by the apparition of two bands (products) and thedisappearance of the initial full-length substrate band. Preferably,said assayed meganuclease variants are digested by proteinase K, forexample, before the analysis of the resulting polynucleotides. Forinstance, the polynucleotide substrate is prepared by the introductionof a polynucleotide comprising the sequence of the target site in aplasmid by TA or restriction enzyme cloning, optionally followed by thelinearization of the plasmid. Preferably, such linearization is not donein the surrounding of the targeted DNA sequence (See Wang et al, 1997,Nucleic Acid Research, 25, 3767-3776; See Examples, Materials & Methods“in vitro activity assays” section) and the characterization papers ofthe initial homing endonucleases.

Alternatively, such in vitro cleavage assay can be performed withpolynucleotide substrates linked to fluorophores, such substratescomprising the targeted DNA sequence. These polynucleotide substratesare immobilized on a solid support. Said solid support is preferably amicroplate (96, 384 or 1536 wells). For example, the polynucleotidescomprising the targeted DNA sequence present a ligand (such as a biotin)at one end, said ligand allowing the immobilization on a solid supportbearing the target of the ligand (for example, streptavidin if biotin isused). The end opposite to the immobilized end is linked to afluorophore. Cleavage leads to loss of fluorescence by release of thefluorochrome from the solid support.

Otherwise, some in vitro cleavage assays can be based on thefluorescence quenching. A fluorophore (for example, FAM or TAMRA) and aquencher (for example, DABCYL) are located on the polynucleotidesubstrate such as the quencher inhibits the fluorescence emission. Thequenching is abolished when the cleavage by the meganuclease variantsoccurs on the polynucleotide substrates. Several examples of thisquenching assays are detailed in Eisenschmidt et al (2002, Journal ofBiotechnology, 96, 185-191) and WO 02/42497, the disclosure of thesedocuments are incorporated herein by reference.

Custom-Made Meganucleases, Polynucleotides Encoding a Custom-MadeMeganuclease, Vectors, Cells and Animals/Plants

The present invention concerns any custom-made meganuclease prepared bythe method according to the present invention and any use of it.Optionally, said meganuclease comprises a purification tag.

The present invention concerns a recombinant polynucleotide encoding acustom-made meganuclease prepared by a method according to the presentinvention. The present invention concerns:

a) any vector comprising a polynucleotide sequence encoding acustom-made meganuclease according to the present invention;

b) any prokaryotic or eukaryotic cell comprising either a polynucleotidesequence encoding a custom-made meganuclease according to the presentinvention or a vector according to a); and,

c) any non-human animal or plant comprising a polynucleotide sequenceencoding a custom-made meganuclease according to the present invention,or a vector according a), or a cell according to b).

As used herein, a cell refers to a prokaryotic cell, such as a bacterialcell, or eukaryotic cell, such as an animal, plant or yeast cell. A cellwhich is of animal or plant origin can be a stem cell or somatic cell.Suitable animal cells can be of, for example, mammalian, avian orinvertebrate origin. Examples of mammalian cells include human (such asHeLa cells), bovine, ovine, caprine, porcine, murine (such as embryonicstem cells), rabbit and monkey (such as COS 1 cells) cells. The cell maybe an embryonic cell, bone marrow stem cell or other progenitor cell.Where the cell is a somatic cell, the cell can be, for example, anepithelial cell, fibroblast, smooth muscle cell, blood cell (including ahematopoietic cell, red blood cell, T-cell, B-cell, etc.), tumor cell,cardiac muscle cell, macrophage, hepatic cell, dendritic cell, neuronalcell (e.g. a glial cell or astrocyte), or pathogen-infected cell (e.g.,those infected by bacteria, viruses, virusoids, parasites, or prions).

The cells can be obtained commercially or from a depository or obtaineddirectly from an individual, such as by biopsy. The cell can be obtainedfrom an individual to whom they will be returned or from individual ofthe same or different species. For example, nonhuman cells, such as pigcells, can be modified to include a DNA construct and then put into ahuman. Alternatively, the cell need to be isolated from the individualfor example, it is desirable to deliver the vector to the individual ingene therapy.

The vector comprising a polynucleotide encoding a custom-mademeganuclease contains all or part of the coding sequence for saidmeganuclease operably linked to one or more expression control sequenceswhereby the coding sequence is under the control of transcriptionalsignals to permit production or synthesis of said meganuclease.Therefore, said polynucleotide encoding a custom made meganuclease iscomprised in an expression cassette. More particularly, the vectorcomprises a replication origin, a promoter operatively linked to saidencoding polynucleotide, a ribosome site, an RNA-splicing site (whengenomic DNA is used), a polyadenylation site and a transcriptiontermination site. It also can comprise an enhancer. Selection of thepromoter will depend upon the desired route for expressing themeganuclease.

The invention concerns a method for producing a custom-made meganucleasecomprising introducing an expression vector into a cell compatible withthe element of said expression vector.

The polynucleotide sequence encoding the custom meganuclease can beprepared by any method known by the man skilled in the art.

Use of the Meganuclease According to the Invention

The custom-made meganucleases according to the present invention are ofgreat utility. Of course, these custom-made meganucleases are preciousfor molecular biology and for genetic engineering, antiviral therapy,genome therapy and gene therapy, more particularly according to themethods described in WO 96/14408, U.S. Pat. No. 5,830,729, WO 00/46385,WO 00/46386, the disclosure of these documents being incorporated byreference.

The custom-made meganucleases with new specificity according to thepresent abolish the limiting step of introducing the recognition andcleavage site for a natural meganuclease in the method of geneticengineering involving meganucleases.

Genetic Engineering and Gene Therapy

The custom-made meganuclease according to the present invention can beused in genetic engineering for the preparation of vector. In vitro,said meganuclease is useful when a rare-cutting endonuclease isnecessary in the vector construction. Said custom-made meganuclease canalso be used for in vivo vector construction. For example, if therecognition and cleavage site for a custom-made meganuclease is locatedon a vector, said meganuclease can be used to induce a homologousrecombination with another vector presenting homology with the sequencesurrounding the cleavage site. Said vector can be a plasmid or a viralvector. Similarly, said custom made meganuclease can be used to delete ahelper vector (generally a plasmid) in a transcomplementing cell linefor the production of retroviruses, AAV or adenoviruses.

Genome engineering is the set of methods used to induce a change in thegenetic program of a living cell and/or organism. The meganucleasesobtained by the method of the present invention allows rational sitedirected modifications of cell genomes. The purpose of these techniquesis to rewrite chromosomes precisely where they should be modifiedleaving the rest of the genome intact. Fields of applications of thegenome engineering are multiple: animal models generation (knock-in orknock-out), protein production (engineering of production strains,protein production in plant and animals for protein production inmilks), agricultural biotechnology (addition or removal of a trait,marker excision), modification and study of metabolic pathway, ortherapy of genetic diseases or viral diseases.

A custom-made meganuclease according to the present invention can beused in a method of genome engineering comprising: 1) introducing adouble-strand break at the genomic locus comprising at least onerecognition and cleavage site of said meganuclease; and, 2) providing atargeting DNA construct comprising the sequence to be introduced flankedby sequences sharing homologies to the targeted locus. Indeed, sharedDNA homologies are located in regions flanking upstream and downstreamthe site of the break in the targeting DNA construct and the DNA thatmight be introduced should be located between the two arms. Saidmeganuclease can be provided directly to the cell or through anexpression vector comprising the polynucleotide sequence encoding saidmeganuclease and suitable for its expression in the used cell.Alternatively, the method of genome engineering comprises: 1)introducing a double-strand break at the genomic locus comprising atleast one recognition and cleavage site of said meganuclease; 2)maintaining under conditions appropriate for homologous recombinationwith the chromosomal DNA homologous to the region surrounding thecleavage site. Any of these methods of genetic engineering could be usedfor repairing a specific sequence, modifying a specific sequence, forattenuating or activating an endogen gene of in for introducing amutation into a site of interest, for introducing an exogenous gene or apart thereof, for inactivating or deleting an endogenous gene or a partthereof, for methylated or demethylating the CpG dinucleotides of agene. The invention relates to the resulting cells and their uses.

The custom-made meganuclease according to the present invention couldalso be used for addition or substitution of telomer, for killing cells,for chromosomic translocation, for changing the chromatinization, or forchromosomic loss.

It is an object of the invention to use at least one custom-mademeganuclease according to the present invention to repair a specificsequence, to modify a specific sequence, to attenuate or activate anendogenous gene of interest, to introduce a mutation into a site ofinterest, to introduce an exogenous gene or a part thereof, and toinactivate or delete an endogenous gene or a part thereof by exposingcells animals, plants to said meganuclease.

Another object of the invention is a composition comprising at least onecustom-made meganuclease according to the present invention. Preferablysaid composition is used for repairing a specific sequence, modifying aspecific sequence, for attenuating or activating an endogenous gene ofinterest, for introducing a mutation into a site of interest, forintroducing an exogenous gene or a part thereof, for inactivating ordeleting an endogenous gene or a part thereof by exposing cells,animals, or plants to said meganuclease. Preferably said compositioncomprises one custom-made meganuclease or two different custom-mademeganucleases. Optionally, said composition can further comprise atargeting DNA construct comprising the sequence to be introduced flankedby homologous sequence to the targeted locus.

More particularly, the invention also relates to the use of a custommade meganuclease obtained by the method according to the presentinvention in a method for treating or prophylaxis of a genetic diseasein an individual in need thereof comprising (a) inducing in cells of theindividual a double stranded cleavage at a site of interest comprisingat least one recognition and cleavage site of said meganuclease, and (b)introducing into the individual a targeting DNA, wherein said targetingDNA comprises (1) DNA sharing homologies to the region surrounding thecleavage site and (2) DNA which repairs the site of interest uponrecombination between the targeting DNA and the chromosomal DNA. Thetargeting DNA is introduced into the individual under conditionsappropriate for introduction of the targeting DNA into the site ofinterest. In a second embodiment the method for treating or prophylaxisof a genetic disease in an individual in need thereof comprises inducingin cells of the individual a double stranded break at a site of interestcomprising at least one recognition and cleavage site of saidmeganuclease under conditions appropriate for chromosomal DNA homologousto the region surrounding to be introduced or deleted into the site ofinterest and repair of the site of interest. Alternatively, cells can beremoved from an individual to be treated, modified by the present methodand reintroduced by autograph into the individual.

The invention relates to custom-made meganuclease obtained by the methodaccording to the present invention in a method for correcting a geneticlesion or abnormality in chromosomal DNA of a cell comprising inducingin the cell double stranded break at a site of interest in the geneticlesion or abnormality comprising at least one recognition and cleavagesite of said meganuclease under conditions appropriate for chromosomalDNA homologous to the region surrounding the site of cleavage to beintroduced into the site of interest and correct the genetic lesion orabnormality. Here, too, the method can be carried out in cells presentin an individual or in cells removed from the individual, modified andthen returned to the individual (ex vivo).

The present invention further relates to the resulting cells and theiruses, such as for treatment or prophylaxis of a condition or disorder inan individual (e.g., a human or other mammal or vertebrate). Forexample, cells can be produced (e.g., ex vivo) by the method describedherein and then introduced into an individual using known methods.Alternatively, cells can be modified in the individual (without beingremoved from the individual).

It is therefore an object of the present invention to use at least onecustom-made meganuclease according to the present invention to prevent,ameliorate or cure a genetic disease by exposing cells, animals orpatients to said meganuclease. Preferably, the invention relates to theuse of one custom-made meganuclease or two different custom-mademeganucleases.

Another object of the invention is a composition comprising at least onecustom-made meganuclease according to the present invention. Preferablysaid composition is used for preventing, ameliorating or curing agenetic disease by exposing cells, animals or patients to saidcomposition. Preferably said composition comprises at least onecustom-made meganuclease. Optionally, said composition can furthercomprise a targeting DNA construct comprising the sequence to beintroduced flanked by sequences homologous to the targeted locus.

Targeting DNA and/or custom-made meganucleases introduced into a cell,an animal, a plant or an individual as described above can be insertedin a vector. Vectors comprising targeting DNA and/or nucleic acidencoding a meganuclease can be introduced into a cell by a variety ofmethods (e.g., injection, transformation, transfection, direct uptake,projectile bombardment, liposomes). Meganucleases can be stably ortransiently expressed into cells using expression vectors. Techniques ofexpression in eukaryotic cells are well known to those in the art. (SeeCurrent Protocols in Human Genetics: Chapter 12 “Vecto Therapy” &Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may bepreferable to incorporate a nuclear localization signal into therecombinant protein to be sure that it is expressed within the nucleus.Custom-made meganuclease can also be introduced into a cell according tomethods generally known in the art which are appropriate for theparticular meganuclease and cell type. Custom-made meganucleasesaccording to the present invention can be introduced into cells usingliposomes or by fusion to the membrane translocating peptide (Bonetta,2002, The Scientist, 16, 38; Ford et al, Gene Ther., 2001, 8, 1-4; Wadia&. Dowdy, 2002, CurrOpin Biotechnol, 13, 52-56).

Once in the cell, the custom-made meganuclease and the vector comprisingtargeting DNA and/or nucleic acid encoding a custom-made meganucleaseare imported or translocated by the cell from the cytoplasm to the siteof action in the nucleus.

Custom-made meganucleases and vectors which comprise targeting DNAhomologous to the region surrounding the cleavage site and/or nucleicacid encoding a custom-made meganuclease can be introduced into anindividual using routes of administration generally known in the art.Administration may be topical or internal, or by any other suitableavenue for introducing a therapeutic agent to a patient. Topicaladministration may be by application to the skin, or to the eyes, ears,or nose. Internal administration may proceed intradermally,subcutaneously, intramuscularly, intraperitoneally, intraarterially orintravenously, or by any other suitable route. It also may in some casesbe advantageous to administer a composition of the invention by oralingestion, by respiration, rectally, or vaginally. The custom-mademeganucleases and vectors can be administered in a pharmaceuticallyacceptable carrier, such as saline, sterile water, Ringers solution, andisotonic sodium chloride solution. Typically, for therapeuticapplications, the custom-made meganucleases will be combined with apharmaceutically acceptable excipient appropriate to a planned route ofadministration. A variety of pharmaceutically acceptable excipients arewell known, from which those that are effective for deliveringmeganucleases to a site of infection may be selected. The HANDBOOK OFPHARMACEUTICAL EXCIPIENTS published by the American PharmaceuticalAssociation is one useful guide to appropriate excipients for use in theinvention. A composition is said to be a “pharmaceutically acceptableexcipient” if its administration can be tolerated by the recipient.Sterile phosphate-buffer saline is one example of pharmaceuticallyacceptable excipient that is appropriate for intravenous administration.The mode of administration is preferably at the location of the targetedcells.

The dosage of custom-made meganuclease or vector of the presentinvention administered to an individual, including frequency ofadministration, will vary depending upon a variety of factors, includingmode and route of administration: size, age, sex, health, body weightand diet of the recipient; nature and extent of symptoms of the diseaseor disorder being treated; kind of concurrent treatment, frequency oftreatment, and the effect desired. For a brief review of pharmaceuticaldosage forms and their use, see PHARMACEUTICAL DOSAGE FORMS AND THEIRUSE (1985) (Hanshuber Publishers, Berne, Switzerland).

For purposes of therapy, the custom-made meganucleases and apharmaceutically acceptable excipient are administered in atherapeutically effective amount. Such a combination is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of the recipient. In the present context, an agent isphysiologically significant if its presence results in a decrease in theseverity of one or more symptoms of the targeted disease or in a genomecorrection of the lesion or abnormality.

In one embodiment of the invention, the custom-made meganucleases aresubstantially non-immunogenic, i.e., engender little or no adverseimmunological response. A variety of methods for ameliorating oreliminating deleterious immunological reactions of this sort can be usedin accordance with the invention. In a preferred embodiment, themeganucleases are substantially free of N-formylmethionine. Another wayto avoid unwanted immunological reactions is to conjugate meganucleasesto polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”)(preferably of 500 to 20,000 daltons average molecular weight (MW).Conjugation with PEG or PPG, as described by Davis et al., (U.S. Pat.No. 4,179,337) for example, can provide non-immunogenic, physiologicallyactive, water soluble endonuclease conjugates with anti-viral activity.Similar methods also using a polyethylene-polypropylene glycol copolymerare described in Saifer et al. (U.S. Pat. No. 5,006,333).

In another use, the custom-made meganucleases can be for in vivoexcision of a polynucleotide fragment flanked by at least one,preferably two, recognition and cleavage site for one or two distinctcustom-made meganucleases. Custom-made meganucleases according to thepresent invention can be used in methods involving the excision oftargeted DNA or polynucleotide fragment from a vector within cells whichhave taken up the vector. Such methods involve the use of a vectorcomprising said polynucleotide fragment flanked by at least one,preferably two, recognition and cleavage site for a custom-mademeganuclease and either an expression vector comprising a polynucleotideencoding said custom-made meganuclease corresponding to the target sitesuitable for the expression in the used cel I, or said custom-mademeganuclease. Said excised polynucleotide fragment can be used fortransgenesis as described in detail in U.S. patent application under No.10/242,664 filed on Feb. 9, 2013. Optionally, said excised targeting DNAcomprises shared DNA homologies located in regions flanking upstream anddownstream the site of the break in the targeting DNA construct and theDNA that might be introduced being located between the two arms. Formore detail see WO 00/46385. Said method of excision of targeting DNAfrom a vector within the cell can be used for repairing a specificsequence of interest in chromosomal DNA, for modifying a specificsequence or a gene in chromosomal DNA, for attenuating an endogeneousgene of interest, for introducing a mutation in a target site, or fortreating or prophylaxis of a genetic disease in an individual.

Antiviral Application

There are currently very few effective anti-viral agents, although themany virally transmitted diseases account for much human suffering andmortality. Thus, there is a great need for safe and effective antiviralagents that can serve in therapies for these diseases, includinglife-threatening and fatal diseases such as hepatitis B and AIDS. 15% ofthe human cancer have viral causes. This rate increases to 80% foruterine and liver cancer. Today, viruses are the third carcinogenicfactors in human.

No existing viral treatment is dedicated to kill the virus infecting thecells and cure the cells out of such infection. The main goal intreating viral infection is reducing viral load in infected cells andwithin a patient. Anti-viral drugs available today are generally toxicand have little specificity. Certain drugs are designed to inhibit acomponent of the virus's replicative machinery such as the enzymesthymidine kinase or reverse transcriptase. These agents do not destroyviral DNA. Other anti-viral agents act to promote the host's immuneresponse so that infected cells are killed more efficiently. Thisresults in non-specific destruction of both the virus and the host cell.

Today, there is a need for new therapeutic agents that specificallydestroy viral DNA without destroying or altering the host cell. Mostviral DNA synthesis occurs within the cell's nucleus; thus it isimportant to generate therapeutic agents that can distinguish betweenthe viral and the host cell DNA.

Sechler (U.S. Pat. No. 5,523,232) disclosed the use of restrictionendonucleases against viruses. This method comprises a step ofadministering to a patient a composition with a restriction endonucleaseable to cleave the targeted virus. The problem of this method is thevery low specificity of the restriction endonuclease (recognition sitesof 4 to 6 nucleotides length) and the high probability to cleave thepatient's genome.

Chandrasegaran (U.S. Pat. No. 6,265,196) disclosed a prospective exampleconcerning the use of a hybrid endonuclease (FoK I domain and zincfinger DNA binding domain) in treatment of viral diseases. However, suchhybrid endonucleases generally lack the ability to specifically act as asingle unique phosphodiester bond or base pair within the DNA targetsite (Smith et al., 1999, supra) and they do not show a high sequencespecificity or any antiviral evidence.

One application of the custom-made meganucleases according to thepresent invention is as therapeutics in the treatment of viral diseasescaused by viruses or retroviruses that present a DNA intermediate.Indeed, many viruses which infect eukaryotic cells possess, during atleast one part of their life cycle, genomes that consist of doublestranded DNA which can be cleaved readily by a meganuclease. Custom-mademeganucleases according to the present invention can be designed so thatthey specifically target viral-specific DNA sequences.

At the opposite of restriction endonucleases and zinc fingers hybridendonucleases, custom-made meganucleases according to the presentinvention present a good specificity for its viral DNA target and theygenerate a unique double strand break only in its viral target. Thisstrategy involves identification of DNA sequences within the viralgenome that are viral-specific, i.e., they are not present within thehuman genome. Once identified, meganucleases that specifically bind andcleave such sequences with high affinity and specificity can be designedusing the method for preparing custom-made meganucleases as described inthe present invention. Then the designed meganucleases are used for thetreatment of viral infection.

It is therefore an object of the present invention to use at least onecustom-made meganuclease according to the present invention to prevent,ameliorate or cure viral infection by exposing the virus and/or infectedcells, plants, animals or patients to these meganucleases. Preferablythe invention relates to the use of one meganuclease or two differentmaganucleases.

Another object of the present invention is to use at least onemeganuclease according to the present invention for inactivating ordeleting virus in biologically derived products and products intendedfor biological uses by treating the products with said meganucleases. Ina particular embodiment, said biological products are blood orblood-derived products. Preferably the invention relates to the use ofat least one meganuclease; optionally two different custom-mademeganucleases.

Another object of the invention is a composition comprising at least onecustom-made meganuclease according to the present invention forpreventing, ameliorating or curing viral infection by exposing the virusor the infected cells, plants, animals or patients to said composition.Preferably, said composition comprises at least one custom-mademeganuclease, optionally two meganucleases.

Another object of the invention, is compositions comprising at least onemeganuclease according to the invention for inhibiting propagation of avirus, inactivating or deleting virus in biologically derived productsor products intended for biological use, or for disinfecting an object.In a particular embodiment, said biological products are blood orblood-derived products. Preferably, said composition comprises onecustom-made meganuclease or two different custom-made meganucleases.

Any virus that contains a double stranded DNA stage in its life cyclecan be targeted for deletion or inactivation by creating a meganucleasethat recognizes DNA sequences specific of the viral genome. Theseviruses could be in replicative or latent form. They could stay eitherepisomal or integrated in the host's genome.

The double-stranded DNA genome viruses are well appropriate to betreated by using meganuclases as defined in the present invention. Amongthem are found the adenoviruses, the herpesviruses, the hepadnaviruses,the papovaviruses, and the poxviruses. Among the herpesviruses are foundherpes simplex virus (HSV), varicella virus (VZV), Epstein-Barr virus(EBV), cytomegalovirus (CMV), herpes virus 6, 7 and 8. Among thehepadnaviruses are found the human hepatitis B virus (HBV). Among thepapovaviruses are found papillomavirus (HPV) (i.e. HPV16 or HPV 18) andpolyoma virus. Among the adenoviruses are found adenovirus 11 and 21which are involved in acute hemorrhagic cystitis. Plants viruses arealso contemplated by the present invention.

The retroviruses are also well appropriate to be treated by usingmeganucleases according to the present invention. Although they are RNAviruses, they are integrated in the host genome as double-stranded DNAform. Among the retroviruses are found the human immunodeficiency virus(HIV) and the human T lymphoma virus (HTLV) (i.e. HTLV1).

Several above-mentioned viruses are well-known to be involved incarcinogenesis: EBV in Burkitt's lymphoma, other lympho-proliferativedisease and nasopharyngeal carcinoma; herpes virus 8 in Kaposi sarcoma;HBV in hepatocellular carcinoma; HPV in genital cancer; HTLV-1 in T-cellleukemia.

For episomal viruses, a double-strand break introduced in its genomeleads to the linearisation of the genome and its degradation. Examplesof episomal viruses are HSV, EBV, and HPV. See example 3.

For integrated viruses, a double strand break introduced in or near theintegrated viral sequence leads to partial or complete deletion of theintegrated viral sequence. Examples of integrated viruses are HPV, HTLV,HBV, and HIV. Several mechanisms could be involved in the deletion. Adouble-strand break in a chromosome induces a gene conversion with thehomologous chromosome, therefore leading to viral sequence deletion. Ifdirected repeat sequences are present near the double strand break, thebreak could also be repaired by SSA (single strand annealing) leading topartial or complete viral deletion. If two double-strand breaks areintroduced, then the chromosome could also be repaired by end joiningleading to partial or complete deletion of the virus, depending on thepositions of the double-strand breaks. See Example 5 in U.S. Pat. No.5,948,678, the disclosure of which is incorporated herein by reference.

To ensure that the targeted viral DNA sequences are not present in thehost's genome, such DNA target sequences should be at least 15nucleotides in length and preferably at least 18 nucleotides in length.As the homing endonuclease present a recognition sequence spanning to12-40 bp, this condition is fulfilled with the custom-made meganucleasesas defined in the present invention. More particularly, I Cre I homingendonuclease has a 22 bp recognition sequence.

Any DNA sequence of viral genomes can be targeted for cleavage bymeganucleases as defined in the present invention. Preferred targetsites include those sequences that are conserved between strains ofvirus and/or which genes are essential for virus propagation orinfectivity. These positions are preferable for at least two reasons.First, essential parts of viruses are less mutated than others.Secondly, it is preferably to target an essential region of the virus tomaximize the inactivation of the virus.

A good target for the custom-made meganuclease could be the viral originof replication (ori) and/or the viral gene encoding an on bindingprotein. Examples of on binding proteins include the HSV-1 UL9 geneproduct, the VZV gene 51 product, the human herpesvirus 6B CH6R geneproduct, the EBV EBNA-1 gene product and the HPV E1 and E2 geneproducts. Other interesting targets for HPV are the genes E6 and E7 asproducts of which are involved in the initiation and maintenance of theproliferative and malignant phenotype. A preferred target is the highlyconserved 62 nucleotides sequence in the pre-core/core region of HPV(E6, E7). Examples of interesting targets for EBV are the genes EBNA andLMP. It could be interesting to target the gene Tax of HTLV-1 whichappears to mediate the oncogenic effects of the virus. For HBV, aninteresting target could be the X gene as the X protein interacts withelements of the DNA repair system and may increase the mutation rate ofp53. For HIV, a preferred target is within TAT, REV, or TAR genes. Theviral targets are not limited to the above-mentioned examples.Optionally, the target DNA could be located in the viral repeatedsequences such as ITR (Inverted Terminal Repeat) and LTR (Long TerminalRepeat).

Preferably, at least two different targeted sites are used. Indeed, asthe main protection of the viruses is their ability to mutate.Therefore, two targeted sites avoid the virus to escape the treatment byusing the custom-made meganucleases, according to the present invention.Moreover, the successive use of different custom-made meganucleases mayavoid the adverse immunologic response. Said different custom-mademeganuclease can present different initial meganucleases, thereforedifferent immunogenicities.

The treatment by custom-made meganucleases according to the presentinvention can be applied either on cells, preferably cells taken fromthe patient, or on the whole body of the patient, with or without organtargeting.

In the case of cell therapy, cells are preferably taken from a patient.These cells are treated by custom-made meganucleases according to thepresent invention in order to inactivate or delete the virus. After thetreatment, cells are reintroduced into the patient. These cells willproliferate and repopulate infected tissues. Preferably, said cells arestem cells, totipotent cells or pluripotent cells. For example, stemcells could be hematopoietic, neuronal, mesenchymal, embryonic,muscle-derived. Other examples of cells are those which are able toregenerate such as the hepatocytes. The treatment by custom-mademeganucleases can be done either by the introduction of meganucleasesinto cells or by the transfection with an expression vector encodingsaid meganucleases. The transfection can be transient or stable. Atransient expression of the custom-made meganuclease allows the cell tobe cleaned up from the virus. A stable transfection allows the cell tobe cleaned up from the virus and avoids a further infection of thetreated cells by the targeted virus.

In the case of a whole therapy, custom-made meganucleases according tothe present invention or expression vector encoding said meganucleasesare introduced into the individual by any convenient means. When thecustom-made meganucleases are introduced or expressed into the infectedcells, the virus is inactivated and/or deleted. The custom-mademeganuclease treatment has no functional impact on healthy cells.

Similarly, such cell or whole antiviral therapy based on the use of atleast one custom-made meganuclease could be used to treat cells ororgans of an animal dedicated to xenotransplantation. The effectivenessof a meganuclease to inhibit viral propagation and infection ispreferably assessed by in vitro and in vivo assays of infection. Suchassays can be carried out first in cell culture to establish thepotential of different meganucleases to cleave a viral DNA in a way thatdeleteriously affects viral propagation. Preliminary studies of thistype are followed by studies in appropriate animal models. Finally,clinical studies will be carried out.

Different viruses require different assay systems, since hosts andculture conditions suitable to different viruses vary greatly. However,such appropriate conditions have been described for culturing manyviruses and these conditions can be used to test the effect of exposingvirus and/or host to meganucleases to determine the ability of theendonuclease to inhibit viral infection. For one discussion of cultureconditions for specific viruses see Chapter 17 in Fields and Knipe,Eds., FIELDS VIROLOGY, 2nd Ed., Raven Press, N.Y. (1990).

A host and/or virus can be exposed at various times during a course ofinfection, under varying conditions, in several amounts, and in avariety of vehicles, to mention just a few relevant parameters that canbe varied, to assess the potential of meganuclease to achieve apotentially therapeutic effect.

In addition, in order to tests ex vivo in cultured cells, potentialtherapeutical meganuclease can be tested in animal models to assessprophylactic, ameliorative, therapeutic and/or curative potential,either alone or in conjunction with other therapeutic agents. In somecases, it will not be possible to culture a virus and it will benecessary to perform all biological assays in animal models. It will bereadily appreciated that different animal models will be appropriate todifferent viruses. Any animal model, however, can be used to assess thetherapeutic potential of a meganuclease.

A potentially effective dose of the assayed meganucleases may beadministered to a suitable population of animals, and the effect of themeganucleases on the course of a viral infection may be assessed bycomparison with an appropriate control. Such methods for assessingpharmacological effect are well known in the art and can readily beadapted to determining the therapeutic profile of the meganucleases.

In one embodiment of the uses according to the present invention, themeganuclease is substantially non-immunogenic, i.e., engender little orno adverse immunological response. A variety of methods for amelioratingor eliminating deleterious immunological reactions of this sort can beused in accordance with the invention. In a preferred embodiment, themeganuclease is substantially free of N-formylmethionine. Another way toavoid unwanted immunological reactions is to conjugate meganucleases topolyethylene glycol (“PEG”) or polypropylene glycol (“PPG”) (preferablyof 500 to 20,000 daltons average molecular weight (MW)). Conjugationwith PEG or PPG, as described by Davis et al., (U.S. Pat. No. 4,179,337)for example, can provide non-immunogenic, physiologically active, watersoluble endonuclease conjugates with anti-viral activity. Similarmethods also using a polyethylene-polypropylene glycol copolymer aredescribed in Saifer et al. (U.S. Pat. No. 5,006,333).

Custom-made meganuclease according to the present invention can beintroduced into cells using liposomes or by fusion to the membranetranslocating peptides (Bonetta 2002, The Scientist, 16, 38; Ford et al,Gene Ther, 2001, 8, 1-4; Wadia & Dowdy, Curr Opin Biotechnol, 13,52-56). Otherwise, meganucleases can be stably or transiently expressedinto cells using expression vectors. Techniques of expression ineukaryotic cells are well known to those in the art. (See CurrentProtocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” &Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may bepreferable to incorporate a nuclear localization signal into therecombinant protein to be sure that it is expressed within the nucleus.

Typically, for therapeutic applications, the custom-made meganucleaseswill be combined with a pharmaceutically acceptable excipientappropriate to a planned route of administration. A variety ofpharmaceutically acceptable excipients are well known, from which thosethat are effective for delivering meganucleases to a site of infectionmay be selected. The HANDBOOK OF PHARMACEUTICAL EXCIPIENTS published bythe American Pharmaceutical Association is one useful guide toappropriate excipients for use in the invention. A composition is saidto be a “pharmaceutically acceptable excipient” if its administrationcan be tolerated by the recipient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable excipient that isappropriate for intravenous administration.

For purposes of therapy, the custom-made meganucleases and apharmaceutically acceptable excipient are administered in atherapeutically effective amount. Said composition can comprise eitherone kind of custom-made meganuclease or several custom-mademeganucleases with different specificity. Such a combination is said tobe administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of the recipient. In the present context, an agent isphysiologically significant if its presence results in a decrease in theseverity of one or more symptoms of a viral illness.

Administration may be topical or internal or other suitable avenue forintroducing a therapeutic agent to a patient. Topical administration maybe by application to the skin, or to the eyes, ears or nose. Internaladministration may proceed intradermally, subcutaneously,intraperitoneally, intraarterially or intravenously, or by any othersuitable route. It also may in some cases be advantageous to administera composition of the invention by oral ingestion, by respiration,rectally, or vaginally. For a brief review of pharmaceutical dosageforms and their use, see PHARMACEUTICAL DOSAGE FORMS AND THEIR USE(1985) (Hans Huber Publishers, Berne, Switzerland).

For topical and internal therapeutic applications, custom-mademeganucleases according to the present invention can be formulated usingany suitable pharmacological technique. For instance, the meganucleasescan be formulated for prolonged release. As described hereinabove,persistence of anti-viral activity of the meganucleases may be increasedor modulated by incorporating the meganucleases into liposomes.

Additionally, the method described in the present invention could beused to modify the physico-chemical properties of meganucleases. Forexample, said method could be used to change the sensitivity of ameganuclease to temperature, pH or salt concentration (e.g. in order todecrease or increase the level at which activity is highest or toenhance the activity at a chosen level), as well as its solubility orstability, or its reaction turnover. Eventually, the method described inthe present invention could also be used to relax or strengthen thespecificity of given meganucleases for preferred DNA sequence targets.

The present invention will be further illustrated by the additionaldescription and drawings which follows, which refers to examplesillustrating the method to produce a custom-made according to theinvention and the use thereof for antiviral therapy and gene therapy. Itshould be understood however that these examples are given only by wayof illustration of the invention and do not constitute in any way alimitation thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses the amino acid sequence of a single chain I-Cre Imeganuclease and one polynucleotide encoding said single chainmeganuclease. In the protein sequence, the two first N-terminal residuesare methionine and alanine (MA), and the three C-terminal residuesalanine, alanine and aspartic acid (AAD). These sequences allow havingDNA coding sequences comprising the NcoI (CCATGG) and EagI (CGGCCG)restriction sites, which are used for cloning into various vectors.

FIG. 2 discloses polynucleotide sequences. FIG. 2A discloses apolynucleotide called “Natural” encoding the I-Cre I homingendonuclease. FIG. 2B discloses a polynucleotide sequence called “Nonhomologous” encoding the I-Cre I homing endonuclease. FIG. 2C disclosesa polynucleotide sequence called “Template” encoding the I-Cre I homingendonuclease comprising the mutation D75NJ. Each I-Cre I homingendonuclease has two additional amino acids (MA) at the N terminal endand three additional amino acids (AAD) at the C-terminal ends. FIG. 2Ddiscloses the polynucleotide sequences of the primers, called UlibIfor,UlibIrev, UlibIIfor, and UlibIIrev, used for the generation of thelibraries UlibI and UlibII.

FIG. 3 is a schematic representation of the polynucleotide sequencecalled “Template” encoding the I-Cre I homing endonuclease comprisingthe mutation D75N. The dark arrows indicate the position of the primersUlibIfor, UlibIrev, UlibIIfor, and UlibIIrev used to generate the twolibraries UlibI and UliblII. D-Helix refers to the LAGLIDADG helix. N75refers to the mutation D75N.

FIG. 4 is a schematic representation of the strategy for the libraryconstruction. Step 1: pET24C-T is a plasmid comprising a polynucleotide<<Template>>. Two PCR amplifications, PCR ulib1 and ulib2, are done witheither UlibIfor and UlibIIrev, or UlibIIfor and UliblIIrev. The PCRulib1 products are cloned in a phagemid pCes4 NHT. The PCR ulib2products are cloned in a plasmid pET24C-T. Step 2: Subcloning a fragmentof Ulib2 vector (pET45C-Ulib2) into the Ulib1 phagemid (pCes4-Ulib1).

FIG. 5: COS cells monolayers were transfected with vector expressing ISee-I (B) or with control plasmid (A). Fourty-eight (48) hours aftertransfection cells were infected with rHSV-1 (30 PFU). Two days latermonolayer was fixed and stained (X-Gal). Infected cells appeared inblue.

FIG. 6: FIG. 6A: cells monolayer was infected with 30 PFU. HSV-1 growthwas quantified by β-galactosidase activity in cell lysate. FIG. 6B, cellmonolayer was infected with 300 PFU. Cell survival was measured byprotein determination in cell lysate. I-Sce refers to vector expressingI-Sce I; I-Sce I(−) refers to a vector in which ORF of I Sce-I wasinserted in reverse orientation; negative control refers to controlplasmid.

FIG. 7: FIG. 7A is a schematic representation of recombinant HSV-1genomic DNA. Cassette containing CMV promoter driving Lac gene wasinserted in the major LAT transcript. I-Sce I restriction site wascloned between promoter and reporter gene, a and b represent primersused for the semi-quantitative PCR. COS-7 monolayers were transfectedwith vector expressing I-Sce I or with control plasmids. Fourty eighthours after transfection cells were infected with rHSV-1 (30 PFU). DNAwas extracted 1, 2 or 3 days after infection. PCR was carried out asdescribed in <<experimental procedures>>. Std refers to Internalstandard; Lac refers to an amplicon of the rHSV-1 Lac gene. I-Sce Irefers to vector expressing I-Sce I; I-Sce I(−) refers to a vector inwhich ORF of I-Sce I was inserted in reverse orientation; negativecontrol refers to control plasmid. FIG. 7B, PCR quantification of theviral thymidine kinase (TK) gene. PCR was carried out at 2 DNAconcentrations. I-Sce I refers to vector expressing I-Sce I; I-Sce I(−)refers to a vector in which ORF of I-Sce I was inserted in reverseorientation; negative control refers to control plasmid.

FIG. 8 illustrates the titration of the virus released in the mediumafter infection of the transfected cells. Every day, medium wascollected and fresh medium was added. Viruses were measured by standardplaque assay. I-Sce I refers to vector expressing I-Sce I; I-Sce I(−)refers to a vector in which ORF of I-Sce I was inserted in reverseorientation; negative control refers to control plasmid.

FIG. 9 represents the I-CreI DNA target and five related targets(C1234=SEQ ID NO: 12; C1221=SEQ ID NO: 13; C4334=SEQ ID NO: 14;H1234=SEQ ID NO: 15; H1221=SEQ ID NO: 16; H4334=SEQ ID NO: 17).Conserved positions are in grey boxes.

FIG. 10 illustrates four binding patterns obtained after screening ofthe Lib2 library with six targets. Positives were identified in a firstscreen and confirmed in a second one during which they were assayedeight times (corresponding to the eight solid bars) on each of thetargets (C1234, C1221, C4334, H1234, H1221 and H4334). Histograms areshown for one clone from each class. Targets are described in FIG. 9.

FIG. 11 illustrates the schematic representation of the target 5vectors. The CpG depleted LacZ gene (LagoZ) is driven by the humanelongation factor 1 alpha promoter. The LagoZ gene is inactivated by theinsertion of I-Sce I cleavage site. Flanking repeats are represented byopen arrows. The length of the homologous sequences are indicated inbold.

FIG. 12 illustrates the effect of the length of homology on single 10strand annealing (SSA) efficiency. Cells monolayers were transfectedwith equimolar amounts of target plasmid bearing different lengths ofhomologous repeat sequences and vector expressing I-Sce I or withcontrol plasmid. Seventy-two hours after transfection cells werecollected and β-galactosidase activity was quantified in cell lysates.(+)I-Sce I, cotransfection with vector expressing I-SceI; (−)I-Sce I,cotransfection with expression vector where the ORF of I-SceI wasinserted in the reverse orientation.

FIG. 13: Cell monolayers were cotransfected with a vector expressing(+)I-Sce I or with a control plasmid (−)I-SceI, Seventy-two hours aftertransfection cells were fixed and stained (X-Gal). FIG. 13A: cells wheregene repair took place appeared in dark. FIG. 13B: frequency of I-SceIinduced recombination on 70 and 220 bp duplication target vectors. Thefrequency is calculated by the ratio of blue cells/transfected cells.

FIG. 14A: X-Gal staining of liver from mice injected with a mixture ofthe target LagoZ gene (30 μg) and an I-Sce I expression vector (10 μg).FIG. 14B: X-Gal staining of liver from mice injected with a mixture ofthe target LagoZ gene (30 μg) and an expression vector where the ORF ofI-SceI was inserted in the reverse orientation (10 μg).

FIG. 15: X-Gal staining of the liver of hemizygote transgenic mice oftwo independent strains infected with the <<Ad.I-SceI>> adenovirus byIV. A. Five days post-infection, β-galactosidase activity is detected inmultiple cells of the entire liver of 10¹⁰ infectious units infected<<58A>> hemizygote. In contrast, no β-galactosidase activity could bedetected by X-Gal staining of the livers of <<Ad.control>>-infectedhemizygote or uninfected <<58A>> littermates (data not shown). B. and C.Fourteen days post-infection, β-galactosidase activity is detected inmultiple cells of the entire liver of 10⁹ infectious units infectedmouse (B) and 10¹⁰ infectious units infected mouse (C). Stronger signalis detected in C compared to B, probably because of the bigger number ofcells that were infected with the <<Ad.I-SceI>>. In contrast, noβ-galactosidase activity could be detected by X-Gal staining of thelivers of uninfected <<361>> littermates (data not shown).

FIG. 16: Fluorescent β-galactosidase assay on liver extract. Twoindependent strains of transgenic mice (58 A and 361) were injected with10⁹ or 10¹⁰ PFU of adenovirus expressing I-SceI (Ad.I-SceI) or controlvirus (Ad.control). Mice were sacrified 5 or 14 days post injection,liver was dissected and proteins were extracted. 30 μl of liver proteinextract were incubated at 37° C. in presence of Fluoresceindigalactoside (FDG). Bars represent the standard deviation of the assay(two measure experiments with samples of the same extracts). NI, noninjected mice; Ad.I-SceI, mice injected with adenovirus expressing I-SceI; Ad.control, mice injected with control adenovirus.

EXAMPLE 1 Single Chain Meganuclease Derived from Dimeric HomingEndonucleases

Some LAGLIDADG homing endonucleases are active as homodimer. Eachmonomer mainly dimerizes through their dodecapeptide motifs. Asingle-chain meganuclease can be engineered by covalently binding twomonomers modified such as to introduce a covalent link between the twosub-units of this enzyme. Preferably, the covalent link is introduced bycreating a peptide bond between the two monomers. However, otherconvenient covalent links are also contemplated. The single-chainmeganuclease preferably comprises two subunits from the same homingendonuclease such as single-chain I-Cre I and single-chain I-Ceu I. Asingle-chain meganuclease has multiple advantages. For example, asingle-chain meganuclease is easier to manipulate. The single-chainmeganuclease is thermo-dynamically favored, for example for therecognition of the target sequence, compared to a dimer formation. Thesingle-chain meganuclease allows the control of the oligomerisation.

A single chain version of I-Crel (scI-CreI) was modeled and engineeredscI-CreI cleaves its cognate DNA substrate in vitro and induceshomolo-gous recombination both in yeast and mammalian cells.

Design of the Single Chain I-CreI Meganuclease

I-CreI from Chlamydomonas reinhardtii is a small LAGLIDADG homingendonuclease that dimerizes into a structure similar to that of largermonomer LAGLIDADG homing endonuclease. To engineer a single chainversion of I-CreI (scI-Crel), two I-CreI copies were fused. Thisrequired placing a linker region between the two domains, and asignificant part of the I-CreI protein had to be removed at the end ofthe domain preceding the linker.

The three-dimensional structure of I-DmoI is comparable to that ofI-CreI, with the exception that I-Dmol comprises a linker region thatleads from one apparent domain to the other. The boundary of that linkerfinely matches related main chain atoms of the I-CreI dimer. In thefirst domain, residues 93 to 95 from the third α-helices of I-CreI andI-DmoI (prior to the linker) are structurally equivalent. At thebeginning of the second LAGLIDADG α-helix (second domain), I-DmoIresidues 104 to 106 correspond to I-CreI residues 7 to 9. In addition,Leu95 and Glu105 from I-DmoI have conserved identities in I-CreI, andI-DmoI residue Arg104 aligns with another basic residue in I-CreI(Lys7). Thus, the single chain I-CreI (scI-CreI), was designed byinserting the I-DmoI linker region from residue 94 to 104 (sequenceMLERIRLFNMR) between a first I-CreI domain (terminated at Pro93) and asecond I-CreI domain (starting at Glu8).

Detailed structural analysis of how the new linker connects the scI-Crelprotein domains (in a modeled structure) revealed no potentialincompatibility. For example, the side chains of nonpolar amino acidstaken from I-DmoI, Met94, Ile98 and Phe109 point inside fitting cavitiesof I-CreI. A single mutation was made (P93A), however, to promoteregularity of the backbone in the α-helix prior to the linker region.(See FIG. 1 for amino acids and polynucleotide sequences).

Materials and Methods

Protein Expression and Purification

His-tagged proteins were over-expressed in E. coli BL21 (DE3) cellsusing pET-24d (+) vectors (Novagen). Induction with IPTG (1 mM), wasperformed at 25° C. Cells were sonicated in a solution of 25 mM HEPES(pH 8) containing protease inhibitors (Complete EDTA-free tablets,Roche) and 5% (v/v) glycerol. Cell lysates were centrifuged twice(15,000 g for 30 min.). His-tagged proteins were then affinity-purified,using 5 ml Hi-Trap chelating columns (Amersham) loaded with cobalt.Several fractions were collected during elution with a linear gradientof immidazole (up to 0.25M immidazole, followed by plateau at 0.5Mimmidazole and 0.5M NaCl). Protein-rich fractions (determined bySDS-PAGE) were concentrated with a 10 kDa cut-off centriprep Amiconsystem. The resulting sample was eventually purified by exclusionchromatography on a Superdex75 PG Hi-Load 26-60 column (Amersham).Fractions collected were submitted to SDS-PAGE. Selected proteinfractions concentrated and dialyzed against a solution of 25 mM HEPES(pH 7.5) and 20% (v/v) glycerol.

In Vitro Cleavage Assays

pGEM plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. or 65° C.in 12.5 mM HEPES (pH 8), 2.5% (v/v) glycerol and 10 mM MgCl2. Reactionswere stopped by addition of 0.1 volume of 0.1 M Tris-HCl (pH 7.5), 0.25M EDTA, 5% (w/v) SDS, and 0.5 mg/ml proteinase K and incubation at 37°C. for 20 minutes. Reaction products were examined following separationby electrophoresis in 1% agarose gels.

Yeast Colorimetric Assay.

The yeast transformation method has been adapted from previousprotocols. For staining, a classic qualitative X-Gal Agarose OverlayAssay was used. Each plate was covered with 2.5 ml of 1% agarose in 0.1M Sodium Phosphate buffer, pH 7.0, 0.2% SDS, 12% Dimethyl Formamide(DMF), 14 mM β-mercaptoethanol, 0.4% X-Gal, at 60°. Plates wereincubated at 37° C.

Mammalian Cells Assays.

COS cells were transfected with Superfect transfection reagentaccordingly to the supplier (Qiagen) protocol. 72 hours aftertransfection, cells were rinsed twice with PBS1× and incubated in lysisbuffer (Tris-HCl 10 nM pH7.5, NaCl 150 mM, Triton X100 0.1%, BSA 0.1mg/ml, protease inhibitors). Lysate was centrifuged and the supernatantused for protein concentration determination and β-galactosidase liquidassay. Typically, 30 μl of extract were combined with 3 μl Mg 100×buffer (MgCl₂100 mM, β-mercaptoethanol 35%), 33 μl ONPG 8 mg/ml and 234μl sodium phosphate 0.1M pH7.5. After incubation at 37° C., the reactionwas stopped with 500 μl of 1M Na₂CO₃ and OD was measured at 415 nm. Therelative β-galactosidase activity is determined as a function of thisOD, normalized by the reaction time, and the total protein quantity.

Results: Single Chain I-CreI Cleaves its DNA Substrate In Vitro and inLiving Cells

A synthetic gene corresponding to the new enzyme was engineered and thescI-CreI protein over-expressed in E. coli. The ability of purifiedscI-CreI to cleave DNA substrates in vitro was tested, using linearizedplasmids bearing a copy of the I-CreI homing site. Similarly to parentI-CreI, the novel enzyme cleaves an I-CreI target site at 37° C.

In order to test the functionality of scl-Crel in vivo, an assay tomonitor meganuclease-induced homologous recombination in yeast andmammalian cells was designed. In yeast, Xenopus oocytes and mammaliancells, DNA cleavage between two direct repeats is known to induce a veryhigh level of homologous recombination between the repeats. Therecombination pathway, often referred to as Single-Strand Annealing(SSA), removes one repeat unit and all intervening sequences. Thus, aSSA reporter vector, with two truncated, non-functional copies of thebacterial LacZ gene and an I-CreI cut site within the interveningsequence was constructed in a yeast replicative plasmid. Cleavage of thecut site should result in a unique, functional LacZ copy that can beeasily detected by X-gal staining.

The reporter vector was used to transform yeast cells. A small fractionof cells appeared to express functional LacZ, probably due torecombination events during transformation. Co-transformation withplasmids expressing either I-CreI or scI-CreI, in contrast, resulted inblue staining for all plated cells. Even in non-induced conditions(glucose), the residual level of protein was enough to induce SSA,suggesting that scI-CreI, as much as I-CreI, is highly efficient inyeast cells. Furthermore, SSA induction was truly dependent on cleavageof the target cut site by I-CreI proteins, as vectors devoid of thatsite display no increase in β-galactosidase activity compared tobackground levels.

The SSA assay was modified for tests in mammalian cells. The promoterand termination sequences of the reporter and meganuclease expressionplasmid were changed, and plasmid recombination was evaluated in atransient transfection assay. Similar levels of induced recombination (2to 3-fold increase) were observed with either scI-CreI or I-CreI. As inthe yeast experiment, recombination depends on an I-CreI cut sitebetween the repeats, for no increase of the β-galactosidase was observedin the absence of this site.

Another recombination assay, based on recombination between invertedrepeats, was also used to monitor meganuclease-induced recombination inCOS cells. As direct repeats can recombine by SSA, homologousrecombination between indirect repeats requires a gene conversion event.Similar stimulation of gene conversion (3 to 4-fold) was observed witheither scI-CreI or I-CreI. As expected for a true homologousrecombination event, no enhancement was observed in the absence of anhomologous donor template.

EXAMPLE 2 Custom-Made Meganuclease Derived from I-Cre I HomingEndonuclease for HIV-2 Target

Construction of a Phage-Displayed Library of I-Cre I Variants

In order to engineer new meganuclease with altered specificities, acombinatorial library was constructed by mutagenesis of the I-Cre Ihoming endonuclease replacing DNA binding residues. Selection andscreening applications then enabled to find those variants that wereable to bind a particular, chosen DNA target. For phage display, asI-Cre I is a homodimer, a phagemid vector was required that encoded twoseparate I-Cre I proteins. Only one of the two I-Cre I copies, which wasfused to the phage coat protein p3, was mutated. The resulting proteinlibrary, in phage display format, comprised thus I-Cre Iwild-type/mutant heterodimers. Eight residues (Q26, K28, N30, Y33, Q38,Q44, R68 and R70) capable together of specific interactions with most ofthe bases in a single hal-site within the DNA target were selected. Ourcombinatorial library was obtained by replacing the eight correspondingcodons with a unique degenerated VVK codon. Eventually, mutants in theprotein library corresponded to independant combinations of any of the12 amino acids encoded by the VVK codon (ADEGHKNPQRST) at eight residuepositions. In consequence, the maximal (theoretical) diversity of theprotein library was 12⁸ or 4.29×10⁸.

Construction of the Library

First, residue D75, which is shielded from solvent by R68 and R70, wasmutated to N (Asn) in order to remove the likely energetic strain causedby replacements of those two basic residues in the library. Homodimersof mutant D75N (purified from E. coli cells wherein it wasover-expressed using a pET expression vector) were shown to cleave theI-CreI homing site. A phagemid vector was then engineered that encodeswild-type I-CreI (FIGS. 2A and 2B: (<<Natural>> or <<Non homologous>>)and the D75N mutant (FIG. 2C: <<Template>>) fused to the phage coatprotein p3 and phage-displayed wild-type/D75N heterodimers were shown tobind that target DNA.

Second, two intermediate libraries of moderate size have been built:Lib1 (residues 26, 28, 30, 33 and 38 mutated; theoretical diversity 12⁵or 2.48×10⁵) and Lib2 (residues 44, 68 and 70 mutated; theoreticaldiversity 12³ or 1.7×10³). DNA fragments carrying combinations of thedesired mutations were obtained by PCR (several reactions in 50 μl),using degenerated primers (FIG. 2D: Uliblfor, Uliblrev, Ulibllfor,Ulibllrev) and as DNA template, the D75N gene. Lib1 and Lib2 wereconstructed by ligation of the corresponding PCR products, digested withspecific restriction enzymes, into the D75N mutant gene, within thephagemid vector and within the pET expression vector, respectively.Digestions of vectors and inserts DNA were conducted in two steps(single enzyme digestions) between which the DNA sample was extracted(phenol:chloroform:isoamylalcohol) and EtOH-precipitated. 10 μg ofdigested vector DNA were used for ligations, with a 5:1 excess of insertDNA. E. coli TG1 cells were transformed with the resulting vectors byelectroporation. To produce a number of cell clones above thetheoretical diversity of either library, up to 35 electroporations ofthe Lib1 ligation samples and 4 electroporations of the Lib2 ligationsamples were necessary. 4×10⁶ (16 times the maximal diversity) and 6×10⁴(35 times the diversity) clones were thus obtained for Lib1 and Lib2,respectively (these numbers were corrected by the number of clonesobtained using ligations done without inserts).

Finally, Lib1 and Lib2 bacterial clones were scraped from plates and thecorresponding plasmid vectors were extracted and purified. The completelibrary was then obtained by sub-cloning a fragment of the Lib2 vectorinto the Lib1 phagemid vector (see FIG. 4 for a schematic diagram of thelibrary construction). Several rounds of DNA 2-step digestions,dephosphorylation, purification, quantification, ligation andelectroporation were performed. After 4 rounds of 150 electroporationshots (which corresponds to 12 ligations of 1.4 μg vector with 0.4 μginsert), 5.5×10⁷ bacterial clones were obtained (after correction forbackground). Bacteria were scraped and stored as a glycerol stock. Inaddition, an aliquot of this glycerol stock was used to inoculate a 200ml culture and the library vector was extracted and purified from thisculture for storage or potential subcloning.

Material and Methods

Protein Expression and Purification

His-tagged proteins were over-expressed in E. coli BL21 (DE3) cellsusing pET 24d (+) vectors (Novagen). Induction with IPTG (1 mM), wasperformed at 15° C. over 5 nights. Cells were cracked for 1 h at 4° C.in a B-Per solution (Bacterial Protein Extraction Reagent, Pierce, 5 mlfor 200 ml culture cell), containing protease inhibitors (CompleteEDTA-free tablets, Roche) and DNase I (80 units)/nuclease (respectively80 and 60 units, Roche). Alternatively, cells were sonicated in asolution of 25 mM HEPES (pH 8) containing protease inhibitors (CompleteEDTA-free tablets, Roche) and 5% (v/v) glycerol.

Cell lysates were centrifuged twice (15,000 g for 30 min). His-taggedproteins were then affinity-purified, using 1 ml Hi-Trap chelatingcolumns (Amersham) loaded with cobalt. Several fractions were collectedduring elution with a linear gradient of immidazole (up to 0.25 Mimmidazole, followed by plateau at 0.5 M immidazole and 0.5 M NaCl).Protein-rich fractions (determined by SDS-PAGE) were concentrated with a10 kDa cut-off centriprep Amicon system. The resulting sample waseventually purified by exclusion chromatography on a Superdex75 PGHi-Load 26-60 column (Amersham).

Fractions collected were submitted to SDS-PAGE. Selected proteinfractions concentrated and dialyzed against a solution of 25 mM HEPES(pH 7.5) and 20% (v/v) glycerol.

In Vitro Cleavage Assay

pGEM plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. in 12.5mM HEPES (pH 8), 2.5% (v/v) glycerol and 10 mM MgCl₂. Reactions werestopped by addition of 0.1 volume of 0.1 M Tris-HCl (pH 7.5), 0.25 MEDTA, 5% (w/v) SDS, and 0.5 mg/ml proteinase K and incubation at 37° C.for 20 minutes. Reaction products were examined following separation byelectrophoresis in 1% agarose gels.

Phagemid Construction

Phage Display of I-Cre I/D75N heterodimer was obtained by using aphagemid harboring two different ORFs as a bicistron, under the controlof promoter pLac. The first one yields a soluble protein fused to aN-terminal signal sequence directing the product into the periplasmicspace of E. coli Gene I-Cre I WT was cloned into this ORF usingrestriction enzymes ApaLI and AscI. The D75N domain was cloned into thesecond ORF using NcoI and Eag I restriction enzyme, leading to a fusionwith the phage coat protein p3 via a hexahis tag, a C-Myc tag and anamber stop codon. This final phagemid was called pCeslCreT. In asuppressive strain like TG1 or XL1blue, and after infection by a helperphage (e.g. M13K07), D75N-p3 fusions are incorporated in the phage coatand the soluble I-Cre I monomers produced in the same compartment willeither dimerize or interact with the displayed D75N domain, therebyproducing particles displaying I-CreI WT/D75N heterodimer.

Phage Production

A 5 mL culture of 2×TY containing 100 μg/ml of ampicillin and 2% glucosewas inoculated with a 1/100 dilution of an overnight culture of bacteriacontaining phagemid pCeslCreT and agitated at 37° C. At an OD₆₀₀ of 0.5,phage helper M13K07 (Pharmacia) was added at a ratio phage:bacteria of20:1. After 30 min at 37° C. without agitation, the culture wascentrifuged for 10 min at 4000 rpm and the pellet was resuspended in 25ml of 2×TY containing 100 μg/mL Ampicillin and 25 μg/ml Kanamycin, andagitated overnight at 30° C. Cultures were centrifuged and supernatantwere used as such in phage ELISA.

PhageELISA

Microtiter plates were coated for 1 h at 37° C. with 100 μl/well ofbiotinylated BSA at 2 μg/mL in PBS. After several washes in PBScontaining 0.1% Tween20 (PBST), wells were incubated with 100 μl/well ofstreptavidin at 10 μg/Ml in PBS and incubated for 1 h at RT. Plates werefurther washed and incubated with biotinylated PCR fragments harboringthe target site, at 250 pM in PBS. After 1 h incubation at RT andwashing, plates were saturated with 200 μl/well of PBS containing 3%powder milk and 25 mM CaCl₂ (PMC). PMC was discarded and plates werefilled with 80 μl of PMC and 20 μl/well of culture supernatantcontaining the phage particles. After 1 h of incubation at RT, plateswere extensively washed with PBST and incubated with 100 μl/well of anti25 M13-HRP conjugated antibody (Pharmacia) diluted 1/5000 in PMC. Plateswere incubated for 1 h at RT, washed and incubated with TMB solution(Sigma). The reaction was blocked with 50 μl/well of 1M H₂SO₄. Plateswere read at 450 nm. A signal higher than 3× the background (irrelevanttarget) can be considered as positive.

PCR-Based Mutagenesis

Plasmid pET24-T45 containing the gene I-CreI D75N was diluted at 1 ng/μlto be used as template for PCR. Degenerated oligonucleotides encodingthe desired randomizations were used to amplify PCR fragments Lib1 andLib2 in 4×50 μl PCR reactions per inserts. PCR products were pooled,EtOH precipitated and resuspended in 50 μl 10 mM Tris.

DNA Digestions

All enzymes and the corresponding buffers were from NEBiolabs.Digestions of up to 10 μg DNA were realised using up to 100 U of a firstrestriction enzyme, at 37° C., in 150 or 500 μl final reaction volume.After 2 h to 6 h, digested DNA was phenol extracted and EtOHprecipitated. Digestion substrates and products were separated usingagarose gel electrophoresis, the desired product being extracted fromthe gel and purified (Nucleospin Extract, Macherey-Nagel). For PCRinserts, digestions were directly purified on Nucleospin columns. Thesecond digestion was then performed in identical conditions. At the endof this second digestion reaction, 0.1 volume of 10×CAP buffer and 0.5μl of CAP were added to the digested vectors, and the samples werefurther incubated for 30 min at 37° C. (The alkaline phosphatase wasinactivated by incubating the sample 10 min at 70° C., after addition ofEDTA). Eventually, the digested and de-phosphorylated DNA was phenolextracted, EtOH precipitated and resuspended in 30 μl of 10 mM Tris pH8.Final DNA concentrations were estimated by comparison of bandintensities in agarose gels after electrophoresis.

Ligations

Large-scale ligations were done at 16° C. (for 16 h) using 1400 ng ofdigested vector and a 5:1 molar excess of digested in 200 μl reactionvolumes and with 4000 U of T4 DNA ligase (NEBiolabs). After ligation,reaction samples were incubated for 20 min at 65° C. to inactivate theligase. The vector DNA was eventually EtOH precipitated and resuspendedat 25 ng/μl in 10 mM Tris pH8.

Electroporations

40 μl of homemade electrocompetent cells TG1 were mixed with 25 ng ofligated DNA (1 μl) in a 2 mm cuvette. After 1 min on ice, cells werepulsed (2.5 Kv, 25 μF, 200 Ohm) and immediately resuspended in 1 ml of2×TY+2% glucose. Cells were placed at 37° C. for 1 h with agitation, andthen plated on large 2×TY plates containing anpicillin (phagemid vector)or kanamycin (pET vector) and 2% glucose and incubated overnight at 30°C. Aliquots were also diluted in 2×TY and plates on small 2×TYAmpicillin glucose plates to obtain isolated colonies allowing thecalculation of library diversities and characterization of severalclones by restriction analysis.

Selection and Screening of Meganuclease Binding to a HIV2-Derived DNATarget from a Library of I-Cre I Variant Using Phage Display

The goal of this project was to obtain a meganuclease capable of cuttinga sequence found in the genome of HIV2 (GGAAGAAGCCTTAAGACATTTTGA). Thehoming endonuclease I-Cre I was used as a scaffold to build a library of10⁸ variants by randomizing 8 residues located at the DNA-bindinginterface of one I-Cre I monomer (see previous section). This librarywas enriched for binders by several rounds of selection/amplificationusing biotinylated DNA fragments harboring the HIV2 derived target (HIV6335). The selected targets were subsequently screened for binding usinga phage ELISA.

Materials and Methods

Phagemid Format

A phagemid based on pCes1 (pCLS346) was chosen. This plasmid harboredtwo different ORFs as a bicistron, under the control of promoter pLac.The first one yielded a soluble protein fused to a N-terminal signalsequence directing the product into the periplasmic space of E. coli. Inour case, this first product was a wild-type monomer of I-CreI. Thesecond ORF encoded an I-CreI monomer that was fused to the phage coatprotein p3 via a hexahis tag, a C-Myc tag and an amber stop codon. In asuppressive strain like TG1 or XLIblue, and after infection by a helperphage (e.g. M13K07), bacteria harboring this phagemid produces phageparticles and around 1-10% of them displays the recombinant protein ontheir surface.

The monomer fused to p3 and randomized on the DNA-binding interface wasincorporated in the phage coat and the soluble I-CreI monomers producedin the same compartment either dimerize or interact with the displayedmonomer, thereby producing particles displaying I-CreI homodimers (orheterodimers if the monomer fused to p3 was mutated).

Target Production

Two complementary primers encoding the desired sequences but harboringan extra adenosine in 3′ were annealed and ligated into pGEM-t Easy(Promega). After sequencing, a correct clone was chosen as template toPCR amplify a biotinylated 200 pb fragment using the kit KOD (Novagen)and primers SP6 (TTTAGGTGACACTATAGAATAC) and biotT7(biot-TAATACGACTCACTATAGG). The PCR product concentration was estimatedon gel and the fragment was used as such in ELISA or selectionprocedures.

Rescue of the Phagemid Library

A representative aliquot of the library (at least 10× more bacteria thanthe library size) was used to inoculate 50 ml of 2×TY containing 100μg/ml ampicillin and 2% glucose (2TYAG) and the culture was agitated at37° C. At an OD₆₀₀ of 0.5, 5 ml of this culture was infected with helperphage K07 at a ratio phage:bacteria of 20:1 and incubated withoutagitation for 30 min at 37° C. After centrifugation at 4000 rpm for 10min at room temperature (RT), the pellet was resuspended in 25 ml of2×TY containing 100 μg/ml ampicillin and 25 μg/ml kanamycin (2TYAK) andagitated overnight at 30° C. The culture was centrifuged at 4000 rpm for20 min at 4° C. and phage particles were precipitated by the addition of0.2 volume of 20% PEG6000/2.5M NaCl for 1 h on ice.

After centrifugation at 4000 rpm for 20 min at 4° C., the phage pelletwas resuspended in 1 ml of PBS and centrifuged at 10 00 rpm for 5 min.0.2 volume of 20% PEG6000/2.5M NaCl was added to the supernatant and themix was centrifuged at 10 000 rpm to pellet the phage particles.Particles were finally resuspended in 250 μl PBS.

Selection Procedure

Phage particles were diluted in 1 ml of PBS containing 3% dry milk and25 mM CaCl (PMC) and incubated for 1 h at RT. 100 pi Streptavidin beads(Dynal, 200 μl for the first round) were washed 3× in PMC and blockedfor Ih in the same buffer. The biotinylated targets were added to thephage at the indicated concentration and the mix was agitated at RT for1 h. Beads were added to the mix and incubated at RT for 15 min. Beadswere collected on the vial wall using a magnet and washed 10× in PMCcontaining 0.1% tween. After a final wash in PBS, beads were resuspendedin 0.5 ml of 100 mM Triethanolamine pH 12 and incubated for exactly 10min. The supernatant were collected and immediately neutralized by 0.5ml of 1 M Tris pH8. An aliquot of this eluate was serially diluted fortitration and with 4 ml 2×TY. 5 ml of exponentially growing TG1 cellswere added and the mix was incubated for 30 min at 37° C. withoutagitation. Cells were plated on large 2TYAG plates and incubatedovernight at 30° C. Colonies were resuspended in 2TYAG, adjusted to anOD₆₀₀ of 100 and kept at −80° C. after addition of 15% glycerol.

Screening by Phage ELISA

Isolated colonies from selection outputs were toothpicked into 100 μl of2TYAG in 96 well plates, and agitated overnight at 37° C. Next day, afresh plate containing 100 μl 2TYAG was isolated using a transferdevice. 50 μl of sterile 60% glycerol was added to the overnight plateand this masterplate was stored at −80° C. The fresh plate was agitatedat 37° C. for 2.5 h, rescued by the addition of 2TYAG containing 2×10⁹pfu of helper phage M13K07, incubated for 30 min at 30° C., spun at 1700rpm for 15 min. Cells pellets were resuspended in 150 μl 2TYAK andagitated overnight at 30° C. After centrifugation, 20 μl of supernatantwas used as described in the previous section.

Results

Selections

Phage particles displaying I-Cre I variants were produced by infectingbacteria harboring the phagemid library with helper phage M13K07. Phageparticles were purified by PEG precipitation and incubated with abiotinylated PCR fragment harboring HIV6335 target. After 1 h ofincubation at room temperature, streptavidin-coated magnetic beads wereadded to the solution to retrieve the biotinylated DMA and bound phages.The beads were extensively washed and the bound phages were eluted by pHshock. Bacteria were infected with the eluted phages and plated on large2×TY plates containing ampicillin and 2% glucose. Serial dilutions of analiquot of the eluted phages were used to infect bacteria to calculatethe number of phage particle and obtain isolated colonies.

The day after, bacteria were scrapped from the large plates and storedas glycerol stocks. An aliquot (representative of the diversity) wasused to produce a new batch of phage particles for a second round ofselection.

The stringency of the selections was increased after each round. Thefirst selection was done using 10 nM of biotinylated target. The secondwas done with 400 pM and the washing steps were extended. The thirdround was done using 250 pM and washed more extensively.

As shown on Table 1, the first and second rounds of selection againstthe HIV2 target lead to an output titer characteristic of backgroundvalues (10⁵ to 10⁶ pfu/ml). However, a significant enrichment wasmeasured on round 3.

TABLE 1 Selection titers. C2H6335: selection done on HIV2 target usingthe library described in the other example. NA: non applicable.Enrichment is defined as (output n + 1/input n + 1)/(output n/input n).Selection Round Input (pfu/ml) Output (pfu/ml) Enrichment 1 6.4 × 10¹¹1.4 × 10⁵ NA 2 4.0 × 10¹² 3.0 × 10⁶  3 3 2.8 × 10¹² 6.9 × 10⁷ 33

Screening by Phage ELISA

80 clones randomly picked from each output (as well as unselectedclones) were used to produce phage particles displaying I-CreI variantsin a monoclonal fashion. Supernatants containing the phage particleswere incubated on 10 biotinylated PCR fragment immobilized on plasticvia streptavidin. Bound phages were stained with an HRP-labeled anti p8(major coat protein) monoclonal antibody (Pharmacia). As shown on Table2, no binders were detected among the unselected clones or from theoutputs of the first round of selection. However 60% of clones pickedafter round 2 against are positive against H6335 but negative on anirrelevant target (P 1234, target of homing endonuclease PI-SceI). Thisresult is in good agreement with the output titer. Indeed this selectiononly resulted in a mild enrichment, suggesting that a large number ofclones still originate from background. As expected, a third round ofselection leads to 99% of strong binders, which explains the largenumber of output phages after this third selection.

TABLE 2 Percentage of positive clones in an ELISA assay directed againstthe I-Crel target (C1234) or the HIV2 derived target (H6335). 77 cloneswere assayed for each output. Round 0: unselected library Selectionround % positive against C1234 % positive against P1234 0 0 0 1 0 0 2 600 3 99 0

Using phage display, new meganucleases were selected from a largelibrary of 1-Cre I variants. Selections on biotinylated DNA targets leadto an increase of output titers characteristic of an enrichment formolecules capable of binding the DNA targets. This enrichment wasconfirmed by phage ELISA. These results demonstrate the efficiency ofthe selection and screening methods.

A Selection/Screen Experiment in Yeast to Identity Novel Meganucleases.Material and Methods Bacterial and Yeast Strains

Every subcloning and plasmid preparations are performed in XLI blue: E.coli provided by Stratagene following standard procedures. Experimentsin S. cerevisiae are done in the following strains:

-   -   FYC2-6A: alpha, trp1Δ63, leu2Δ1, his3Δ200    -   FYBL2-7B: a, ura3Δ851, trp 1Δ63, Ieu21Δ, lys2Δ202    -   YASP3 (derived from FYC2-6A): alpha, ura3::SSA-ura3-HIV2-KanR,        ade2::SSA-ade2-HIV2-TRP1, trp 1Δ63, leu2Δ, his3Δ200

PLASMIDS

-   -   pCLS0279: ADH1 promoter, TRP1 selectable marker and ARS-CEN        origin of replication, β-galactosidase SSA target, HIV2 6335        cleavage site.    -   pCLS0569: kanamycin resistance cassette, HIV2 6335, internal        fragment of the URA3 gene.    -   pCLS0570: kanamycin resistance cassette, HIV2 6335, internal        fragment of the LYS2 gene.    -   pCLS0576: TRP1 selectable marker, HIV2 6335, internal fragment        of the ADE2 gene.    -   pCLS0047: Galactose inducible promoter, LEU2 selectable marker        and 2 micron origin of replication.

Results

An in vivo assay in yeast that allows to screen mutagenized I-Crelprotein variants with detectable activity towards a specified target wasperformed.

A library of mutated I-Crel meganucleases has been first selected by aphage display procedure, resulting in a sub-library enriched forvariants of interest, able to bind the HIV2 6335 target. The insertsfrom this enriched sub-library are subcloned into pCLS0047 under thecontrol of a galactose-inducible promoter, for further selection inyeast. However, we can produce the library directly in the suitableyeast expression vector, and void the phage display step.

A specific yeast strain (YASP3) containing two reporter systemsintegrated in chromosomes was prepared. These two reporter systems arebased on recombination by Single Strand Annealing (SSA). SSA is inducedby specific cleavage of the HIV2 6335 site.

Namely, a URA3 SSA target and an ADE2 SSA target were introduced. TheURA3 SSA target was a modified ura3 gene with 2 direct repeats of 600base pairs separated by 4.3 kb (containing a kanamycin resistancecassette and the HIV2 6335 cleavage site). The strain was unable to growon a minimal medium lacking uracile but was resistant to G418. When thistarget was cleaved and recombined properly, the yeast was able to growon media without uracil and was sensitive to G418.

The ADE2 SSA target was a modified ade2 gene with 2 direct repeats of1.1 kb separated by 3.6 kb (containing a tryptophan selectable markerand the HIV2 6335 cleavage site). Because of this mutated ade2 gene, theyeast strain was unable to grow on a minimal medium lacking adenine, butharbored a red color on a medium with a low adenine content. Because ofthe tryptophan selectable marker, it was able to grow on minimal mediawithout tryptophan. When this target was cleaved and recombinedproperly, the yeast was white, able to grow on media without adenine andunable to grow on a minimal medium lacking tryptophan.

Basically, the recipient yeast strain was red (on low adenine medium),G418 resistant, tryptophan prototroph and auxotroph for uracile andadenine. If a specific meganuclease is expressed in this strain andcleaves its target sites, the resulting yeast clone is white, G418sensitive, prototroph for tryptophan and auxotroph for uracile andadenine.

The YASP3 strain was validated by determining the level of spontaneousrecombination of each target alone and of both targets taken together.The URA3 SSA 10 target recombined spontaneously as an uracileprototrophe, G418 sensitive at an approximate 6 10⁻⁴ rate. The ADE2 SSAtarget recombined spontaneously as an adenine prototrophe at anapproximate 2.7 10⁻³ rate. Recombination of both markers occurredspontaneously (resulting in uracile/adenin rototrophes) at anapproximate 10⁻⁶ rate.

A pilot experiment with 1.5×10 in transformants showed no backgroundlevel of uracileladenine prototrophes means that the number of falsepositive clones should be less than 10 after a transformation experimentwith a library that would yield about a million of independent clones.

The library is used to transform YASP3. A classical chemical/heat chockprotocol that routinely gives 10⁶ independent transformants per pg ofDNA was used (Gietz, R. D. and Woods, R. A., 2002) Transformation ofyeast by lithium acetate/single-stranded carrier DNA/polyethylene glycolmethod (Methods Enzymol, 350, 87-96).

Transformation of the strain with the library gives more than 10⁶independent yeast transformants from which a number of clones are ableto grow on a selective medium without uracile, leucine and containinggalactose as a carbone source and a low amount of adenine. Among thoseclones, the interesting ones are white indicating that they contain aLEU2 vector allowing the expression of a meganuclease specific for HIV26335 site and that the enzyme is able to cut both URA3 and ADE2reporters.

The positive clones are isolated and screened for their ability toinduce the specific recombination of a plasmidic SSA β-galactosidasetarget (pCLSO279). This plasmidic reporter was a modified LacZ gene with2 direct repeats of 825 base pairs separated by 1.3 kb (containing aURA3 selectable marker and the HIV2 6335 cleavage site). The vector(which can be selected on a medium without tryptophan) is used totransform a yeast strain (FYBL2-7B) and clones are maintained on minimalmedia 35 lacking uracile to maintain the unrecombined LacZ target.

Yeast clones resulting from the selection experiment are mated with theyeast strain containing the SSA β-galactosidase target. Diploids areselected and assayed for induced β-galactosidase activity. A number ofclones are expected to behave as false positives at this step. Theycorrespond to the background level of spontaneous recombination of theURA3 and ADE2 SSA targets. All remaining clones (uracile and adenineauxotrophes able to induce recombination of the SSA LacZ target) aretrue positives expressing a meganuclease cleaving the HIV2 6335 targetin vivo. Also, other experiments, based on the ones described above, canbe used to determine more precisely the activity of such novel enzymes.

EXAMPLE 3 Use of Meganuclease for Antiviral Therapy

Experimental Procedures

Cells

COS-7 cell lines from the American Type culture collection (ATCC) werecultured in DMEM plus 10% fetal bovine serum. PC-12 cells from ATCC weregrown in RPMI1640 supplemented with 10% heat-inactivated horse serum and5% heat-inactivated fetal bovine serum. PC-12 cells were differentiatedas previously described (Su et al., 1999, Journal of Virology,4171-4180). Briefly, cells were seeded on 6 well-plate at 5 10⁴ cellsper well. The following day, cells were incubated in PC-12 mediumcontaining 100 ng/ml of 2,5S NGF (Invitrogen). Medium was changed everythree days. After 7 days of incubation, undifferentiated cells wereeliminated by adding 2 μM of fluorodeoxyuridine (FdUrd).

Construction of Recombinant HSV-1

HSV-1 was purchased from ATCC. Viruses were propagated on COS-7 cells atlow MO1 (0.01 PFU/cell). Recombinant virus (rHSV) were generated aspreviously described (Lachmann, R. H., Efstathiou, S., 1997, Journal ofVirology, 3197-3207). A 4.6 Kb pstI-bamHI viral genomic DNA fragment wascloned in pUC 19. Based on HSV-1 sequence from database (ID: NC 001806),this region represents nucleotides 118867 to 123460. A cassetteconsisting of a CMV promoter driving Lac gene expression was introducedinto a 168 bp HpaI deletion. This region is located within the major LATlocus of HSV. I-Sce I cleavage site was finally cloned directly afterthe CMV promoter. This construct was used to generate recombinantviruses. Plasmid was linearized by XmnI digestion, and 2 μg of thisplasmid DNA was contransfected with 15 μg of HSV-1 genomic DNA preparedfrom COS-7 infected cells by CaCl₂ method. After 3 or 4 days, infectedcells were harvested and sonicated. Aliquot of the lysed cells were usedto infect COS monolayer. Virus recombinant were selected by overlayingCOS monolayer with 1% agarose in medium containing 300 μg/ml of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Blue clones werepicked and further subjected to three rounds of plaque purification.Presence of the I-Sce I site was confirmed by PCR and in vitro I Sce-Ienzymatic digestion.

Viral Inhibition

6 well-plate were seeded with 2.10⁴ cells per well. The next day COS-7cells were transfected with 0.5 μg of plasmid expressing ISce-I orcontaining the ISce-I ORF in the opposite orientation by the. EFFECTENEmethod according to the manufacturer protocol. We achieved routinely inour laboratory 60 to 70% efficiency using this methodology. Forty-eighthours later, subconfluent transfected cells were infected with rHSV. Forinfection, rHSV-1 was diluted in PBS containing 1% fetal bovine serumand adsorbed onto cells for 20-40 min at 37° C., in humidified incubatorwith 5% CO₂.6 wells-plates were infected at 30 or 300 PFU per well forrespectively viral inhibition or cells survival experiments. Cells wereharvested at day 1, 2, and 3 and β-galactosidase activity was assayedand DNA extracted.

β-Galactosidase Activity

Cell monolayer was fixed in 0.5% glutaraldehyde in 100 mM PBS containing1 mM MgCl₂ at 4° C. for 10 minutes. After one wash with detergentsolution (100 mM PBS, 1 mM MgCl₂, 0.02% Nonidet p-40) cells wereincubated at 37° C. in X-Gal stain solution (10 mM PBS, 1 mM MgCl₂, 150mM NaCl, 33 mM K₄Fe(CN)₆.3H₂O, 33 mM K₃Fe(CN)₆, 0.1% X-Gal) until colordevelopment. Beta-galactosidase activity was also measured on cellextract with o-nitrophenyl-β-D-galactopyrannoside (ONPG) as substrate.Cell monolayer was washed once with PBS. Cells were then lysed with 10mM Tris ph 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitors. After30 minutes incubation on ice cell lysate was centrifuged andβ-galactosidase was assayed. Typically 30 μl of supernatant was combinedwith 270 μl of reaction buffer (10 mM PBS; ph 7.5, 1 mM MgCl₂, 0.3%(β-mercaptoethanol) containing 800 μg/ml ONPG. The reaction was carriedout at 37° C. and stopped with 0.5 ml of 1M NaCO₃. Optical density wasmeasured at 415 nm. Beta-galactosidase activity is calculated asrelative unit normalized for protein concentration and incubation time.

Semi-Quantitative PCR

To measure viral replication of rHSV, oligonucleotides were designed toamplify a 217 bp fragment from Lac gene. The standard DNA used in thisassay was generated by cloning this fragment in a Bluescript plasmid,and by inserting a 50 bp fragment downstream to the 5′ oligonucleotide.PCR of the standard produced 267 bp amplicon. Series of PCR (not shown)were carried out to fix the amount of standard and DNA sample, and thenumber of cycles to achieve linear response of the amplification. Thebasic semi-quantitative PCR were carried out in a total volume of 30 μl,using the READYMIX™ TAQ (Sigma) with 20 pmols of each primers and 180 pgof DNA. The tubes were heated for 4 min at 94° C. and subjected to 22cycles: 94° C. for 1 min, 62° C. for 50 sec, 72° C. for 2 min, and 72°C. for 7 min.

Virus Titration

In one series of experiments, the culture medium was collected every dayat days 1, 2, 3, and 4, and fresh medium was added. In the other, themedium was not changed during experiment and aliquots were collectedevery day. To monitor for the release of HSV-1 progeny, aliquot ofmedium were titred on COS-7 cells by standard plaque assay.

Results

The effect of I-Sce I on viral replication was examined using arecombinant Herpes simplex virus carrying a I-Sce I restriction site(rHSV). For convenience, rHSV-1 was build with a cassette containing CMVpromoter driving the Lac gene. I-Sce I site was inserted at the junctionof the CMV promoter and Lac gene.

The expression cassette was cloned by homologous recombination in themajor LAT locus which allowed Beta-galactosidase (β-gal) expressionduring lytic infection in COS-7 cell monolayer. Strikingly transfectionof I-Sce I expression vector before viral infection virtually completelyinhibited HSV-1 plaque formation in COS cells (FIG. 5) as shown by X-Galcoloration. In contrast, control transfection with expression vectorcontaining I-Sce I open reading frame in the reverse orientation whichdid not allow any functional transcript, did not affect viralreplication. Furthermore, 48 hours after infection, the cells werechecked for I-Sce I expression. All the lysis plaques formed in cellsmonolayer transfected with I-Sce I expression vector represented cellswhich did not expressed I-Sce I (the transient transfection is about 70%efficient). However, cells expressing I-Sce I surrounding the lysisplaque inhibited the viral propagation. The I-Sce I effect was confirmedby measuring the β-galactosidase activity in a cell lysate. Afterinfection of COS-7 cells monolayer transiently expressing I-Sce I with30 Pfu per well cell monolayer was collected at day 1, 2, and 3 postinfection and β-gal was assayed. FIG. 6A shows a drastic decrease of theβ-galactosidase activity reflecting the inhibition of rHSV-1replication. The protective effect of I-Sce I over a time course ofrHSV-1 infection was evaluated next. At 3 days after infection, cellstransfected with I-Sce I expressing vector shown no sign of cytopathiceffect whereas control cultures were completely lysed as shown in FIG.6B. In an effort to quantify the degree of inhibition of viral DNAreplication by I-Sce I, we have set-up a semi-quantitative PCR. GenomicDNA was extracted from cells at day 1, 2, and 3 after infection. PCR wascarried out with primers a and b (FIG. 7A) generating a 217 bp ampliconin Lac gene. An internal standard was added in sample before PCR toquantify DNA. Lac gene was virtually not detectable in I-Sce Iexpressing cells at 3 days post-infection (FIG. 7A). In contrast cellsthat did not receive I-Sce I expression vector shown high levels ofvirus DNA. This result was confirmed by PCR using primers in viralendogenous gene (FIG. 7B). Amplification of Thymidine Kinase (TK) geneshown that I-Sce I inhibited the viral replication. Finally COS-7 cellsexpressing active I-Sce I or I-Sce I ORF in the reverse orientation wereinfected with rHSV-1 and the concentration of virus released in themedium at different time points was measured by plaque assay (FIG. 8).Viruses were quantified in a rough array at day one when I-Sce I wasproduced. Viruses production was still markly decreased two days afterthe infection when compared with cells which did not express I-Sce Ishowing that I-Sce I effectively inhibited viral replication. Thiseffect was still observed at day three although in a lesser extent.Probably the high mutation rate occurring during viral replicationallowed emergence of mutant HSV-1 which were able to escape the I-Sce Iactivity.

Taking together, these results demonstrates that I-Sce I and moregenerally meganucleases can be used to inhibit viral infection. The useof custom made meganuclease or combination of custom-made meganucleasesdesigned to cut specific viral sequences could represent a powerful newstrategy in the antiviral therapy.

EXAMPLE 4 Meganuclease with Altered Binding Properties Derived fromI-Crel Homing Endonuclease

The purpose of this experiment was to obtain novel meganucleases bindingtarget sites close to the I-Crel natural target site. A series of 6targets were used (FIG. 9), including the wild-type natural I-Creltarget (named C1234), the HIV2 target described in example 2 (named hereH1234), and four additional targets. These four additional targets are24 bp palindromes corresponding to inverted repeats of a 12 bp halfI-CreI or HIV2 target site: C1221 and C4334 are inverted repeats of thefirst half and second half, respectively, of the C1234 target; H1221 andH4334 are inverted repeats of the first half and second half,respectively, of the H1234 target. In contrast with example 2, themethod used here did not involve any selection step, but was based onthe extensive screening of the Lib2 library (see example 2). Threeresidues (Q44, R68 and R70) capable of base specific interactions withthe DNA target were selected. The combinatorial library was obtained byreplacing the three corresponding codons with a unique degenerated VVKcodon. Eventually, mutants in the protein library corresponded toindependent combinations of any of the 12 amino acids encoded by the VVKcodon (ADEGHKNPQRST) at three residue positions. In consequence, themaximal (theoretical) diversity of the protein library was 12³ or 1728.

Materials and Methods Construction of a Phage-Displayed Library ofI-Crel Variants.

First, residue D75, which is shielded from solvent by R68 and R70, wasmutated to N (Asn) in order to remove the likely energetic strain causedby replacements of those two basic residues in the library. Homodimersof mutant D75N (purified from E. coli cells wherein it wasover-expressed using a pET expression vector) were shown to cleave theI-CreI homing site. A phagemid vector was then engineered that encodesthe D75N mutant (FIG. 2C: <<Template>>) fused to the phage coat proteinp3 and phage-displayed D75N monomers were shown to bind the I-CreInatural DNA target (C1234 on FIG. 9).

Then, DNA fragments carrying combinations of the desired mutations wereobtained by PCR (several reactions in 50 μl), using degenerated primers(FIG. 2D: UlibIIfor, UlibIIrev) and as DNA template, the D75N gene. Lib2was constructed by ligation of the corresponding PCR products, digestedwith specific restriction enzymes, into the D75N mutant gene, within thephagemid vector, as described in example 2.

Screening of Meganucleases Binding to the 6 Different Targets

Screening was performed by Phage ELISA, as described in example 2.

Results

4560 clones (more than 2.5 times the theoretical mutant librarydiversity) were individually picked and screened by phage ELISA with the6 different targets. 28 positives (clones binding one of the sixtargets) were identified. For validation, these 28 clones werere-assayed by phage ELISA, 8 times in parallel with the 6 differenttargets; 20 clones were thus confirmed as true positives. Finally, all28 clones were sequenced.

TABLE 3 Sequence of the proteins found in the four different classes.Only amino acids from position 44, 68 and 70 are indicated. Clones foundtwice are labeled with (2). Class I Class II Class III Class IV QRK(NTQH)N QRT (2) Unknown QRR QRN (RG)(ED) sequence H(KEQ)E QRA QQK (2)QSR QNK QTR (2) QQR QHK DSH Unknown sequence

Four different patterns (ELISA results) could be observed. FIG. 10features one representative example for each one. The first class (ClassI) corresponds to a strong binding of C1234, C1221, C4334 and H4334. Thewild-type protein (QRR) was recovered in this class, showing that ClassI profile is the regular binding profile of the original scaffold. Twovariants were also shown to display such binding (QRK and another yetnot completely identified mutant).

Variants from the second class have lowered their affinity for alltargets, but H4334, since no binding was observed with C1234, C1221 andC4334. Eight different proteins were found to belong to this class, plusa protein which sequence could not be determined. Among the sequencevariants of Class II, five retain the Q44 amino acid from the wild-typesequence, and one of the two arginines in position 68 or 70. However, inone mutant (DSH), none of the amino acids from position 44, 68 and 70has been retained. Class III (4 different proteins) has a more complexpattern, as it retains apparent binding for the C1221 and H4334 target.Finally, one protein (Class IV) retains only a slight binding for targetC1221 as none of the other targets are bound anymore.

It is difficult to draw conclusions from Class IV, since the residualbinding with C1221 is very low, and sequencing of the unique Class IVmutant has failed. However, comparison of Class II and III with thewild-type profile of Class I clearly shows that the binding specificityhas been altered.

The conclusion is that even from small libraries such as Lib2(complexity 1.7 10³), variants with altered binding profiles can beisolated, as shown in FIG. 10. Therefore, strategies based on screening,starting with larger mutant libraries, should allow the identificationof more dramatic alterations, for instance binding for targets that werenot bound by the initial protein scaffold. In addition, this approachleads to the identification of many different proteins for each profile.An extensive study of this kind should also bring the basis of a betterunderstanding of DNA/meganuclease interactions.

EXAMPLE 5 Comparison of Selection and Screening Methods in Yeast Library

The purpose is here to compare the screening and selection methods inyeast. Whereas screening is the extensive examination of each individualclone of a population for its desired properties (for us, the cleavageproperties), selection is an enrichment step: an initial library issubmitted to the selection process, resulting in a sublibrary enrichedfor clones with the desired properties.

Since the throughput of the screening process can be insufficient toprocess very large number of clones, one or several selection steps canbe useful when one has to deal with a very high diversity. However,selection can bring several unexpected bias, resulting in the selectionof other properties than the ones wished by the operator. Thus, it isextremely important to validate any selection process carefully.

Therefore, a selection method and a screening method were developed tolook for meganuclease cleaving specific DNA target in yeast. Both arebased on the production, by homologous recombination, and moreprecisely, by Single-Strand annealing, of specific markers, uponcleavage of the DNA target by the meganuclease within the yeast cell.The principle of these assays is described in Example 2. Selection isbased on the restoration of an auxotrophy marker, (URA3 in Example 2,ADE2 and LYS2 in this example), whereas screening is based on therestoration of a color marker, (LacZ and ADE2 in example 2), LacZ inthis example (since an ade2 mutation results in no growth, or in a yeastred color, depending on the amount of adenine in the culture medium, itcan be used for both selection and screening).

Thus, the clones screened as positive with and without selection on asmall library were compared, in order to check whether the selectionmethod is suitable.

Material and Methods Bacterial and Yeast Strains

Every subcloning and plasmid preparations are performed in XL1-20 blue:E. coli provided by Stratagene following standard procedures.

Experiments in S. cerevisiae are done in the following strains, whereincutI-CreI represents the cleavage site for I-CreI:

FYC2-6A: alpha, trp1Δ63, leu2Δ1, his3Δ200

FYBL2-7B: a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202

YAP4 and YDD6 (derived from FYC2-6A): alpha,lys2::SSA-ura3-cutI-CreI-KanR, ade2::SSA-ade2-cutI-CreI-TRP1, trp1Δ63,leu2Δ1, his3Δ200

Plasmids

pCLS050: ADH1 promoter, TRP1 selectable marker and ARS-CEN origin ofreplication of β-galactosidase SSA target, I-CreI cleavage site.

pCLS0047: Galactose inducible promoter, LEU2 selectable marker and 2micron origin of replication.

Results

A library of mutated I-CreI meganucleases, namely Lib2 (see example 4)was introduced into a yeast vector (pCLS0047).

Two specific yeast strain (YAP4 and YDD6) containing two reportersystems integrated in chromosomes were prepared. These two reportersystems are based on recombination by Single Strand Annealing (SSA). SSAis induced by specific cleavage of the I-CreI site.

Namely, a LYS2 SSA target and an ADE2 SSA target were introduced. TheLYS2 SSA target was a modified lys2 gene with 2 direct repeats of 3240bp base pairs separated by 4.3 kb (containing a kanamycin resistancecassette and the I-CreI cleavage site). The strain was unable to grow ona minimal medium lacking lysine but was resistant to G418. When thistarget was cleaved upon overexpression of I-CreI and recombinedproperly, the yeast was able to grow on media without lysine and wassensitive to G418.

The ADE2 SSA target was a modified ade2 gene with 2 direct repeats of1.1 kb separated by 3.6 kb (containing a tryptophan selectable markerand the I-CreI cleavage site). Because of this mutated ade2 gene, theyeast strain was unable to grow on a minimal medium lacking adenine.Because of the tryptophan selectable marker, it was able to grow onminimal media without tryptophan. When this target was cleaved andrecombined properly, the yeast was able to grow on media without adenineand unable to grow on a minimal medium lacking tryptophan.

Basically, the recipient yeast was G418 resistant, tryptophan prototrophand auxotroph for both lysine and adenine. If a specific meganuclease isexpressed in this strain and cleaves the I-Cre1 target sites, theresulting yeast clones are G418 sensitive, auxotroph for tryptophan andprototroph for lysine and adenine.

The Lib2 library was used to transform YAP4 and YDD6. A classicalchemical/heat chock protocol that routinely gives us 10⁶ independenttransformants per μg of DNA was used (Gietz and WQoods, 2002, MethodsEnzymol, 350, 87-96).

Transformation of the strain with the library gives more than 10⁶independent yeast transformants on a selective medium without leucine(selection for the meganuclease expression vectors), and containingglucose as a carbone source and a low amount of adenine. Such clones canbe screened after mating with the yeast strain containing the SSAβ-galactosidase target (FYBL2-7B transformed with pCLS050). Diploids areselected and assayed for induced β-galactosidase activity. Screening of2400 independent clones resulted in the identification of 15 positives.DNA from these clones was recovered, electroporated into E. coli, forrecovery of the meganuclease expression plasmid. For each of these 15positives, two E. coli clones were amplified and sequenced. Nodifference of sequence was observed between two E. coli clones obtainedfrom the same positive yeast clone. Plasmids were then retransformedinto the yeast YAP4 or YDD6 strain, and screening was done again. Itconfirmed the results of the primary screen for the 15 different yeastclones.

The same screening experiment was then performed, with the exceptionthat a selection step was added. The Lib2 library was transformed intoYDD6, and the cells were plated onto a selective medium without leucine(selection for the meganuclease expression vectors), and containingglucose as a carbone source. After three days of growth, colonies wereresuspended in water, and plated onto a selective medium without leucine(selection for the meganuclease expression vectors), adenine and lysine(selection for the clones wherein the two ADE2 and LYS2 SSA reporter hadrecombined) and containing galactose as a carbone source. 960 cloneswere obtained, and 845 (88%) were screened as positive after mating withthe yeast strain containing the SSA β-galactosidase target (FYBL2-7Btransformed with pCLS050). Eighteen out of them were confirmed by asecond round of screening.

These results gave a measure of the enrichment in a single round ofselection. Since screening gave us 15 positives out of 2400 cloneswithout selection and 845 out of 960 with selection, the enrichment is(845/960)/(15/2400)=140.

Then the 33 confirmed clones (18 obtained with and 15 obtained withoutselection) were sequenced. The table below describes the clones obtainedby selection and screening. Since Lib2 results from mutation of residuesQ44, R68 and R70 of I-CreI, clones are described by three letters,corresponding to the amino acids (one letter code) presents at positions44, 68, and 70. For example, wild type would appear as QRR, as a TRRmutant corresponds to a single mutation at position 44, replacing theglutamine with a threonin.

TABLE 4 Comparison of the clones obtained either by simple screening orselection and screening. Number of clones Number of clones containingthe mutation containing the mutation Mutant I-CreI after simplescreening of after selection AND Protein 2500 clones screening TRR 6clones (out of 15) 12 clones (out of 18) QRA 3 clones (out of 15)  5clones (out of 18) QAR 2 clones (out of 15) TRL 1 clone (out of 15) TRA1 clone (out of 15) TRN 1 clone (out of 15)  1 clone (out of 18) ARN 1clone (out of 15)

Clearly, the selection process is very efficient, since an enrichmentfactor of 140 is observed after selection. Among the positives, clonesTRR and QRA are the most frequent with and without selection. Foursequences were found after simple screening, and not after selection.However, these differences are not statistically significant: only 18 ofthe 845 positives found after selection were sequenced andcharacterization of a larger sample should identify more differentpositive variants.

These results show that with a small library such as Lib2, positives canbe selected efficiently, whereas the biases that could be introduced bythis selection system are small if any. Thus, this selection procedurecan be used to enrich larger libraries for variants of interest, forexample when these libraries cannot be entirely processed by thescreening method. In this example, the mutants that have still theability to cleave the I-CreI target site were recovered. However,mutants cleaving novel targets could be obtained just in the same way.

EXAMPLE 6 Screening of Active Meganuclease Based on their CleavageProperties In Vitro, and in Mammalian Cells

The purpose of this experiment is to demonstrate that active andinactive meganucleases can be distinguished on simple screening assaysin vitro and in mammalian cells. The in vitro assay is similar to arestriction assay, and the assay in mammalian cells is based oncleavage-induced recombination. Cleavage in mammalian cells is similarto the assay in yeast (example 2): cleavage induced recombination (andmore precisely single-strand annealing) results in a functional LacZreporter gene which can be monitored by standard methods. However, incontrast with the yeast assay, which relies on stable replicativeplasmids, the cell-based assay is working with transient matrix.

As for examples 4 and 5, the Lib2 library was used. Three residues (Q44,R68 and R70) capable of base specific interactions with the DNA targetwere selected. The combinatorial library was obtained by replacing thethree corresponding codons with a unique degenerated VVK codon.Eventually, mutants in the protein library corresponded to independantcombinations of any of the 12 amino acids encoded by the VVK codon(ADEGHKNPQRST) at three residue positions. In consequence, the maximal(theoretical) diversity of the protein library was 12³ or 1728. Thetarget used was the natural I-CreI target (named C1234).

2000 clones were individually picked, and tested with the two screeningmethods, to establish a comparison of these methods. Positives were thensequenced and analyzed.

Materials and Methods

Construction of a Library of I Crel Variants for Expression In Vitro.

First, residue D75, which is shielded from solvent by R68 and R70, wasmutated to N (Asn) in order to remove the likely energetic strain causedby replacements of those two basic residues in the library. Homodimersof mutant D75N were shown to cleave the I-CreI homing site. The librarywas constructed in the pTriex vector (Novagen).

Then, DNA fragments carrying combinations of the desired mutations wereobtained by PCR (several reactions in 50 μl), using degenerated primers(FIG. 2D: UlibIIfor, UlibIIrev) and as DNA template, the D75N gene. Lib2was constructed by ligation of the corresponding PCR products, digestedwith specific restriction enzymes, into the D75N mutant gene, within thepTriex vector, as described in example 2.

Production of Meganucleases In Vitro

50 ng of DNA plasmid solution in 2 μl were mixed to 4 μl of RTS solution(Rapid Translation System RTS 100, E. coli HY kit from Roche). Proteinproductions were done in vitro at 30° C. for at least 4 h. Afterproduction of the proteins, the fresh preparation was diluted indistillated water prior to trying the meganuclease activity.

In Vitro Cleavage Assay

pGEMT plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. in 12.5mM HEPES (pH 8), 2.5% (v/v) glycerol and 10 mM MgCl2. Reactions werestopped after 1 hour by addition of 0.1 volume of 0.1 M Tris-HCl (pH7.5), 0.25 M EDTA, 5% (w/v) SDS, and 0.5 mg/ml proteinase K andincubation at 37° C. for 20 minutes. Reaction products were examinedfollowing separation by electrophoresis in 1% agarose gels.

Cleavage in Mammalian Cells

CHO cells were cotransfected with the meganuclease expressing pTriExplasmid and the reporter plasmid. The reporter plasmid contains aninactive LacZ gene under the control of an appropriate promoter. LacZ isinactive because it contains an internal duplication of 220 bp, and aninsertion of the target site (24 to 80 pb), located between the two 220bp repeats. For transfection, Polyfect transfection reagent was usedaccordingly to the supplier (Qiagen) protocol. 72 hours aftertransfection, tissue culture medium was removed and cells were incubatedin lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton X100 0.1%,BSA 0.1 mg/ml, protease inhibitors). The whole lysate was combined with0.1 volume of Mg 100× buffer (MgCl₂ 100 mM, β-mercaptoethanol 35%), 1.1volume of ONPG 8 mg/ml and 7.8 volume of sodium phosphate 0.1 M pH7.5.After incubation at 37° C., the reaction was stopped with 0.5 volume of1 M Na₂CO₃ and OD was measured at 415 nm. The relative β-galactosidaseactivity is determined as a function of this OD. Positives are cloneschoosen by comparison with a negative control where an empty expressionvector is tranfected to the cells. Their β-galactosidase activities ishigher than (M+2 E) (where M is the average β-galactosidase activity ofthe negative controls and E the standard deviation between those samemeasurements).

Results

2000 clones (more than the theoretical mutant library diversity) wereindividually picked and cultured in 96 deep-well plates. Plasmid DNA wasextracted using a BioRobot8000 platform (Qiagen) with the Qiaprep 96Turbo BioRobot kit (Qiagen). Since the pTriEx plasmid has been designedto drive the expression of protein in bacteria and mammalian cells,plasmid DNA was then used for both the in vitro assay and the assay incells. All the positives obtained with either method were rechecked forcleavage in vitro and in cells.

For screening with the in vitro assay, meganuclease were produced invitro from each individual plasmid with the RTS (Roche) system, and thentested for cleavage of a linearized pGEMT (InvitroGene) plasmidcontaining the targets. The digests were then run on an electrophoresisgel to detect the expected cleavage products.

For screening with the assay in mammalian cells, the plasmid werecotransfected with reporter plasmid containing the target sites in CHOcells, and cleavage-induced recombination was monitored 72 hours later,as a function of β-galactosidase activity.

Out of the 2000 clones, 206 clones (10.3%) were found to be positivewith the in vitro assay. In mammalian cells, 85 clones (4.3%) were foundto be 20 positives. Out of these 291 clones, 82 were positive in bothmethods.

121 different variant proteins were identified after sequencing ofpositive clones. The identity and cleavage properties of these 121variants are shown in the table below. 54 variants display a detectablecleavage activity in both assays, as 66 are positives only for cleavagein vitro. A single variant is positive in the cell-based

25 assay but not in vitro.

TABLE 5 Sequence end cleavage properties of the different variants Invitro Residue 44 Residue 68 Residue 70 cleavage SSA in cells Ala ArgAla + + Ala Arg Arg + + Ala Arg Gly + + Ala Arg His + + Ala Arg Ser + +Ala Lys Lys + + Ala Thr Lys + + Asn Arg Arg + + Asn Arg His + + Asn ArgPro + + Asp Pro Thr + + Gln Ala Arg + + Gln Ala His + + Gln Arg Ala + +Gln Arg Arg + + Gln Arg Asn + + Gln Arg His + + Gln Arg Pro + + Gln ArgSer + + Gln Arg Thr + + Gln Asn Arg + + Gln Gln Arg + + Gln Gln Thr + +Gln His Arg + + Gln His Asn + + Gln His Gln + + Gln Lys Ala + + Gln LysGln + + Gln Lys Lys + + Gln Lys Thr + + Gln Ser Gly + + Gln Ser His + +Gln Thr Arg + + Glu Arg Arg + + Pro Arg Gly + + Pro Arg Thr + + Ser ArgAsn + + Ser Arg Lys + + Ser Lys Arg + + Ser Thr Arg + + Thr Ala Arg + +Thr Ala Thr + + Thr Arg Ala + + Thr Arg Arg + + Thr Arg Asn + + Thr ArgGly + + Thr Arg Lys + + Thr Arg Ser + + Thr Arg Thr + + Thr Gln Arg + +Thr Gly Arg + + Thr Lys Arg + + Thr Thr Arg + + Thr Thr Lys + + Ala AlaArg + − Ala Arg Asn + − Ala Arg Thr + − Ala Gln Arg + − Ala Gln Lys + −Ala Gln Asn + − Ala Gly Arg + − Ala His Arg + − Ala Ser Lys + − Ala ThrArg + − Arg Ala Ser + − Arg Arg Asn + − Arg Thr Asn + − Asn Ala Arg + −Asn Arg Asn + − Asn Arg Gly + − Asn Arg Ser + − Asn Arg Thr + − Asn AsnArg + − Asn Gln Arg + − Asn Thr Arg + − Asp Arg Lys + − Asp Ser Asp + −Gln Ala Ala + − Gln Ala Ser + − Gln Ala Thr + − Gln Asn Asn + − Gln AsnGly + − Gln Asn Pro + − Gln Asn Thr + − Gln Gln Asn + − Gln Lys Asp + −Gln Pro Asn + − Gln Ser Ala + − Gln Ser Asn + − Gln Thr Ser + − Gly AlaArg + − Gly Arg Ala + − Gly Arg Arg + − Gly Arg Gly + − Gly Arg Ser + −Gly Arg Thr + − Gly Gln Arg + − Gly Gln Lys + − Gly His Arg + − His AlaArg + − His Arg Ala + − His Arg Thr + − Lys Ala Ser + − Lys Ala Thr + −Lys Arg Gln + − Lys Arg Thr + − Pro Asn Thr + − Ser Arg Ala + − Ser ArgGly + − Ser Arg His + − Ser Arg Thr + − Ser Asn Arg + − Ser His Arg + −Ser Ser Arg + − Ser Ser Lys + − Thr Arg Asp + − Thr Arg His + − Thr GlnLys + − Thr His Arg + − Thr Ser Arg + − Gln His His − +

These results clearly show the discrimination of positive and negativeclones can be achieved in two different assays, based on cleavage invitro and cleavage in mammalian cells. Cleavage in vitro seems to bemore sensitive, since much more positives were identified. Thesedifferences can be due to many factors resulting either frommeganuclease expression, or activity, either from the detection based onrecombination. However, all but one clone (Gln44 His68 His70) identifiedin cells appeared to be confirmed in vitro, which shows that thecell-based assay is reliable to identify functional endonucleases.

EXAMPLE 7 Meganuclease-Induced Recombination of an ExtrachromosomalReporter In Toto Using I-Sce I Expressing Plasmid A—Optimization of theReporter System

Experimental Procedures

Vectors Construction

The target vectors are based on a LagoZ expression vector driven bypromoter of the human EF 1-alpha gene. This promoter has been shownpreviously to have wide expression spectrum in vivo (Kim, D. W.,Uetsuki, T., Kaziro, Y., Yamaguchi, N., Sugano, S, 1990, Gene, 91,217-223). The promoter region includes splice donor and acceptor sitesin the 5′ untranslated region of the h-EF 1-alpha gene-LagoZ is a CpGisland depleted LacZ gene designed to abolish gene silencing intransgenic mice (Henry I, Forlani S, Valliant S, Muschler J, Choulika A,Nicolas J F, 1999, C R Acad Sci III. 322, 1061-70). To construct targetvectors with different lengths of homology, the 3′ fragment of the LagoZgene was first deleted (about 2000 bp) and replaced by the I-Sce Icleavage site. The 3′ fragments of different lengths were generated bydigestion of the parental plasmid. These fragments contained differentamounts of homology with the 5′ fragment of the LagoZ gene. Finallythese DNA fragments were individually cloned adjacent to the I-SceIcleavage site, creating different target vectors with 0, 70, 220, 570,and 1200 bp of homology, respectively.

Cell Culture

COS-7 and CHO-K1 cell lines from the American Type Culture 10 Collection(ATCC) were cultured in DMEM or Ham's F 12K medium respectively plus 10%fetal bovine serum. For I-Sce I induced Single Strand annealing (SSA)assays, cells were seeded in 12 well-plates at a 15.10³ cells per wellone day prior transfection. Transient transfection was carried out thefollowing day with 500 ng of DNA using the EFFECTENE transfection kit(Qiagen). Equimolar amounts of target plasmid and I-Sce I expressionvector were used. The next day, medium was replaced and cells wereincubated for another 72 hours.

β-Galactosidase Activity

Cell monolayers were fixed in 0.5% glutaraldehyde in 100 mM PBScontaining 1 mM MgCl₂ at 4° C. for 10 minutes. After one wash withdetergent solution (100 mM PBS, 1 mM MgCl₂, 0.02% Nonidet p-40) cellswere incubated at 37° C. in X-Gal stain solution (10 mM PBS, 1 mM MgCl₂,150 mM NaCl, 33 mM K₄Fe(CN)₆.3H₂O, 33 mM K₃Fe(CN)₆, 0.1% X-Gal) untilcolor development. Beta-galactosidase activity was also measured in cellextracts with o-nitrophenyl-β-D-galactopyrannoside (ONPG) as asubstrate. Cell monolayers were washed once with PBS. Cells were thenlysed with 10 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, proteaseinhibitors. After 30 minutes incubation on ice, cells lysates werecentrifuged and β-galactosidase was assayed. Typically 30 μl ofsupernatant was combined with 270 μl of reaction buffer (10 mM PBS; pH7.5, 1 mM MgCl₂, 0.3% β-mercaptoethanol) containing 800 μg/ml ONPG. Thereaction was carried out at 37° C. and stopped with 0.5 ml of 1M NaCO₃.Optical density was measured at 415 nm. Beta-galactosidase activity iscalculated as relative unit normalized for protein concentration andincubation time.

Results

When a DNA double-strand break (DSB) is introduced between two repeatedsequences, it induces homologous recombination resulting in a deletionof the repeats, together with all the intervening sequences. Therecombination pathway is often referred to as the single-strandannealing (SSA) pathway. A reporter system was designed to monitormeganuclease-induced SSA in animal models in toto. In order to optimizethe reporter system, the correlation between meganuclease-induced SSAefficiency and repeat length was first examined. Different targetvectors carrying a LagoZ gene containing duplications of various sizeswere constructed (FIG. 11). The presence of the duplication and of theI-Sce I cleavage site inactivates the gene. The repair of the LagoZ geneby SSA results in the loss of one repeat and of the cleavage site, andin the restoration of a functional LagoZ gene. LagoZ codes for theβ-galactosidase enzyme which can be detected by colorimetry. Transienttransfection with equimolar amounts of target vector and I-SceIexpression vector or expression vector that doesn't express themeganuclease were carried out in CHO or COS-7 cells. The resultsobtained with the different constructs are presented in FIG. 12. I-Sce Iinduced DSBs clearly stimulate the SSA repair mechanism. Furthermore,homology of 70 bp was sufficient to achieve nearly maximum efficienciesof induced SSA, while the level of spontaneous recombination (withoutI-Sce I induced DSB) was minimal. With duplication of 220 bp maximumefficiency was achieved while no additional gains in SSA efficiency wereobserved with longer duplications. Similar results were obtained withCOS-7 cells (data not shown). 70 and 220 bp of homology gave the bestratio of activity vs background. Because β-galactosidase is assayed incell lysates and one single cell can contain several copies of thetarget plasmid, it is impossible to evaluate the absolute SSA efficiencyby this method. Therefore direct coloration of the cellular monolayerwas performed 72 hours post-transfection (FIG. 13). Virtually no bluecells were detected in the absence of the meganuclease (FIG. 13A). Incontrast, many β-galactosidase-positive cells are present when I-Sce Iis cotransfected with the target vector, demonstrating the stimulationof homologous recombination by meganuclease induced DSB. The efficiencyof I-Sce I induced SSA was calculated by counting the blue cells (cellswhere recombination has taken place) and comparing it with the number oftransfected cells (cells that effectively received DNA). FIG. 13B showsthat 50 to 60% of the cells undergo homologous recombination when I-SceI is present along with the target vector carrying 70 or 220 bpduplications while spontaneous recombination represents less than 0.1%of the events. Thus, the construct with the 70 bp and 220 bp of homologyas well as the transgene were selected for the animal study.

B. Meganuclease-Induced Recombination of an Extrachromosomal Reporter InToto

Experimental Procedures

Hydrodynamic-Based Transfection In Vivo

Transduction of the mouse liver cells was performed by hydrodynamic tailvein injections as previously described (Zhang, G., Budker, V., Wolff,A., 1999, Human Gene Therapy, 10, 1735-1737; Liu, F. Song, Y. K., Liu,D., 1999, Gene Therapy, 6, 1258-1266). This method allows efficienttransduction and expression of exogenous genes in animals byadministration of plasmid DNA by tail vein injection. Briefly, DNA ismixed in 1.5 to 2 ml of PBS, which represents 10% of the animal'sweight. Tail vein injections are subsequently performed with a 26-gaugeneedle over a 5-10 sec period using sterile materials and workingconditions. Using such a protocol, almost exclusively liver cells aretransduced, thus the I-SceI-mediated SSA event leading to the correctionof the LagoZ gene was studied in the liver. The I Sce I expressingvector used is the pCLS 197 corresponding to the I-SceI-coding sequences(U.S. Pat. No. 5,474,896) under the control of the CMV promoter in a pUCbackbone and is 5737 bp long.

OF1 mice weighing fifteen to twenty grams were obtained from CharlesRiver Laboratories, France. A total of twenty micrograms of DNA,containing equal amounts of target vector and either an I-SceIexpression or control vector, was injected into mouse tail veins. Thetarget vector contains the LagoZ gene interrupted by an I-Sce I cleavagesite flanked by direct repeat sequences containing 70 bp of homology.Control mice were injected with a mixture of the target vector and aplasmid that does not express I-Sce I.

β-Galactosidase Activity

Three days after injection, mice were euthanized by cervical dislocationand X-Gal stainings of their livers were performed. Livers weredissected out of the animals in cold Ix PBS and the lobes were cut inpieces of about one fourth a centimeter in order to allow a betteraccess of the X-Gal in the tissue. Then liver pieces were placed infresh cold PBS 1× in a 12-well cell culture plate kept on ice, and fixedin 4% paraformaldehyde for 1 hour under agitation at 4° C. Samples werethen washed 3 times at room temperature for 30 minutes with wash buffer(100 mM sodium phosphate pH=7.3, 2 mM MgCl₂, 0.01% sodium deoxycholate,0.02% NP-40 by volume). In toto X-Gal staining was performed overnightat 37° C. in staining solution (5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide, 1 mg/ml X-gal, 20 mM Tris pH=7.3 in washbuffer). Finally samples were washed extensively with PBS and examinedunder microscope. Pictures were taken with a Nikon Coolpix camera undera Nikon SMZ 1500 binocular.

Results

Cellular study has shown that homology of 70 bp is sufficient to achievenearly maximal efficiencies of DSB induced SSA, while the level ofspontaneous recombination (without I-SceI induced DSB) is minimal. Thus,in a first attempt to stimulate recombination in vivo, transientexperiments were performed. A mixture of the target vector (30 μg) andeither the I-SceI expression or control plasmid (10 μg) were introducedinto the liver via a hydrodynamic tail vein injection method. FIG. 12shows a magnified picture of liver collected and stained 3 days afterinjection. Blue dots represent cells where a defective LagoZ gene,bearing an I-SceI site flanked by a 70 bp duplication, was repaired.After meganuclease induced DSB, the SSA pathway results in the deletionof one repeat and reconstitution of a functional gene. Activeβ-galactosidase encoded by the LagoZ gene can then be detected by X-Galstainings. Furthermore, no gene correction was detected in the absenceof the meganuclease expression vector. These data represent the firstevidence that meganuclease induced recombination can be stimulated inliver and that in toto repair of an extrachromosomal target can beachieved.

EXAMPLE 8 Meganuclease-Induced Recombination of a Chromosomal ReporterIn Toto Using I-See I Expressing Adenovirus

In order to demonstrate meganuclease-induced genomic surgery of achromosomal reporter in toto in different mice tissues, the repair ofthe LagoZ gene in toto was tested by transducing cells of several organswith an I-SceI-expressing adenovirus, <<Ad.I-SceI>>. Control transgeniclittermates were infected with a non-I-SceI-expressing adenovirus,<<Ad.control>>. Adenovirus infections in transgenic mice were performedby intravenous (IV) injections. Repair of the LagoZ gene in toto inseveral tissues was then tested by two methods that detectβ-galactosidase activity in toto, X-gal staining and FDG assays.

Experimental Procedures

Transgenic Mice

The transgene used for the generation of transgenic founders was aBglII/NotI fragment of 5919 bp carrying the defective LagoZ gene,inactivated by a LagoZ duplication of 70 bp or 220 bp and the I-SceIcleavage site, under the control of the human elongation factor 1 alphapromoter (see FIG. 11).

Transgenic founder were generated by classical transgenesis, i.e. bymicroinjecting the linear and purified BglII/NotI fragment describedabove at 500 copies/picolitres into the male pronuclei of fertilized ovaat the one-cell stage derived from the mating of B6D2F1 males andfemales purchased from Elevage Janvier. Microinjections were performedunder a Nikon TE2000 microscope with Normarski DIC with eppendorftransferMan NK2 micromanipulators and eppendorf Femtojet 5247micro-injector. After injections, ova were transferred to surrogatepseudopregnant B6CBAF1/J females (Elevage Janvier) for development anddelivery. Transgenic mice generated by this procedure were identified byPCR and Southern Blot analysis on genomic DNA extracted from tailbiopsies of F0 mice. The molecular characterization of the transgeneintegration was done by PCR and Southern Blot analysis.

Then the founder was mated to B6D2F1 mice in order to obtain hemizygotetransgenic F1 animal. Expression of the transgene was tested byperforming an RT-PCR experiment on RNAs extracted from a tail biopsyfrom a transgenic F1 animal using Qiagen RNeasy kit (cat No. 74124).Hemizygote F1 mice were then mated to B6D2F1 mice in order to establishan F2 hemizygote transgenic strain.

Two independent strains were used bearing either 220 bp or 70 bp longLagoZ gene repeated sequences. These transgenic strains are referred toas strain <<361>> and <<58A>>, respectively.

The molecular characterization of the transgene integration showed thatthe integration is about 5 direct repeats of the BglII/NotI transgene in<<361>> and 2 inverted repeats plus 5 direct repeats in <<58A>>.Hemizygote mice were identified by tail biopsies, genomic DNA extractionand PCR analysis. <<361>> and <<58A>>, hemizygote mice were then usedfor in toto I-Sce I mediated-LagoZ gene repair and transgeniclittermates were used as negative controls.

Adenovirus-Based Transduction In Toto

Recombinant type V adenovirus bearing the I-SceI meganuclease codingregion under the control of a CMV promoter, <<Ad.I-SceI>> was providedby Q BIO gene company at 1.58 10¹¹ infectious units concentration scoredby the TCID₅₀ method. The negative adenovirus control <<Ad.control>> wasas well provided by Q BIO gene company at 3.76 10¹¹ infectious unitsconcentration. Recombinant type V adenovirus infections were performedby intravenous (IV) injections in transgenic mice tail veins. Transgenicmice were weighed and anesthetized before infections by intraperitonealinjection of a mixture of Xylasin (100 mg/kg) and Ketamine (10 mg/kg).IV infections were performed with 10¹⁰ infectious units/animal in avolume of 400 μl. Infections were performed in 4 to 7 weeks-oldtransgenic mice. Adenovirus infected mice and uninfected controllittermates were bred in isolator until sacrificed for β-galactosidaseassays.

β-Galactosidase Activity

Adenovirus-infected mice were sacrificed by CO₂ inhalation from 5 to 14days-post-infections (dpi) and their organs were processed forβ-galactosidase assays. About 10% of the liver (8 mm³) was employed forprotein extraction and the remaining 90% was used for β-galactosidase intoto X-gal assays (protocol described previously).

Fluorescent β-galactosidase assays were incubated at 37° C. in 96 wellplates. The assays were performed in a total volume of 100 μl containing30 μl of protein extract, 1 μM Fluorescein digalactoside (FDG, Sigma),0.1% β-mercaptoethanol, 1 mM MgCl₂, and 100 mM Phosphate buffer, pH 7.0.The plates were scanned on the Fluoroskan Ascent (Labsystem) at5-minutes intervals. The β-galactosidase activity is calculated asrelative unit normalized for protein concentration and incubation time.

Results

Two <<58A>> transgenic mice were 1V-injected with 10¹⁰ infectious unitsof <<Ad.I-SceI>> adenovirus in order to target a DSB in-between the 70bp duplicated LagoZ sequences and induce the repair of the reportergene. At various times post-injection the mice were sacrificed andseveral organs were dissected and analyzed by in toto X-gal assays. Bluestaining was detected as dispersed cells over the entire liver ofinfected mouse euthanized at 5 dpi (FIG. 15A). No staining could bedetected in the other organs tested, i.e. kidneys, spleen, heart andlungs. Two <<58A>> transgenic mouse littermates were used as controls,one IV-injected with 10¹⁰ infectious units of the control adenovirus<<Ad.control>> and the other uninfected. No β-galactosidase activitycould be detected in the liver of either control (data not shown).Similar results were obtained with two <<361>> transgenic mice injectedwith 10⁹ and 10¹⁰ infectious units of the Ad.I-SceI adenovirus (FIGS.15B and 15C respectively). These results were confirmed by measuring theβ-galactosidase activity in liver extract (FIG. 16). A high activity wasdetected in liver of mice injected with Adenovirus expressing I-SceI(Ad. I-Sce-I). In contrast, Non-injected mice (NI) shows only a residualbackground activity similar to the activity detected in mice injectedwith the control adenovirus (Ad.control).

The IV-injected mouse with 10¹⁰ infectious units of <<Ad.1-SceI>>adenovirus exhibited more stained liver cells and more β-galactosidaseactivity than the IV-injected mouse with 10⁹ infectious units of<<Ad.1-SceI>> adenovirus. These results suggest that I-SceI-inducedrecombination could be dose dependent and that a better yield of I-SceI-induced recombination could be obtained by increasing theinjected-adenovirus titer. Thus, I-SceI-induced genome surgery should bedetectable in other organs reported to be less sensitive to type Vadenovirus infection.

Taken together, these data strongly suggest that the reporter generepair was induced by the activity of the I-SceI meganuclease. Thisresult is the first evidence that I-SceI and more generally themeganucleases can be used in toto to induce efficient site-specifichomologous recombination leading to the repair of a chromosomal gene.Thus, this result opens applications in the field of gene therapy inmammals.

1-40. (canceled)
 41. A method for producing LAGLIDADG meganucleasevariants derived from an initial LAGLIDADG meganuclease, saidmeganuclease variants being able to cleave a desired DNA target which isdifferent from the DNA target of the initial LAGLIDADG meganuclease,wherein said method comprises: a) generating a combinatorial library ofgenes coding for LAGLIDADG meganuclease variants by introducingdiversity into several codons of a gene encoding said initial LAGLIDADGmeganuclease, wherein said codons code for amino acid residues of thefour-stranded beta-sheet of one domain or one subunit of said initialLAGLIDADG meganuclease that interact directly or indirectly withdifferent bases of the DNA target of said initial meganuclease; b)screening meganuclease variants encoded by the genes of thecombinatorial library obtained in a) by an in vivo cleavage assay in anisolated yeast cell or an isolated mammalian cell, under conditionswhere a double-strand break in the DNA target which is generated by saidvariants leads to the activation of a positive selection marker or areporter gene, or the inactivation of a negative selection marker or areporter gene, by recombination-mediated repair of said DNAdouble-strand break, and c) isolating the genes encoding themeganuclease variants obtained in b) which are able to cleave said DNAtarget.
 42. The method according to claim 41, wherein said amino acidresidues that interact directly or indirectly with the DNA target of theinitial meganuclease are selected from the group consisting of: D, E, H,K, N, Q, R, S, T and Y.
 43. The method according to claim 42, whereinsaid amino acid residues are substituted with an amino acid selectedfrom the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 44.The method according to claim 41, wherein said initial LAGLIDADGmeganuclease is a natural or a modified meganuclease.
 45. The methodaccording to claim 41, wherein said initial LAGLIDADG meganuclease isselected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, andPI-Pfu I.
 46. The method according to claim 45, wherein said initialLAGLIDADG meganuclease is I-Cre I.
 47. The method according to claim, 46wherein I-Cre I variants are prepared by introducing amino acidsubstitutions in at least three positions of the I-CreI of SEQ ID NO:10, wherein said positions are selected from the group consisting ofQ26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10.
 48. Themethod according to claim 47, wherein said I-Cre I variants compriseamino acid substitutions at the following positions Q26, K28, N30, Y33,Q38, Q44, R68 and R70.
 49. The method according to claim 47, whereinsaid I-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q44, R68 and R70.
 50. The method accordingto claim 47, wherein said I-Cre I variants comprise amino acidsubstitutions at the following positions Q26, K28, Y33, Q38, Q44, R68and R70.
 51. The method according to claim 47, further introducing anamino acid substitution with an uncharged amino acid, at position 75 ofthe I-Cre I of SEQ ID NO:
 10. 52. The method according to claim 51,wherein said uncharged amino acid is an asparagine residue.
 53. Themethod according to claim 41, wherein in said in vivo cleavage assay,said isolated yeast or mammalian cell comprise an expression cassettecomprising said desired DNA target and the coding region of an activenegative selection marker, the coding region of an inactive positiveselection marker, or the coding region of an active or inactive reportergene product.
 54. The method according to claim 41, wherein in said invivo cleavage assay, said isolated yeast or mammalian cell comprises anexpression cassette comprising (i) said desired DNA target and apolynucleotide comprising two direct repeats of an internal portion of apolynucleotide encoding an inactive positive selection marker, whereinthe two repeats are separated by the desired DNA target, or (ii) saiddesired DNA target and a polynucleotide comprising two direct repeats ofan internal portion of an inactive reporter gene, wherein the tworepeats are separated by the desired DNA target.
 55. The methodaccording to claim 53, wherein in said in vivo cleavage assay, saidisolated yeast or mammalian cell comprise an expression cassettecomprising said desired DNA target and (i) a polynucleotide encoding aninactive positive selection marker due to the presence of a mutation ora deletion, or (ii) an inactive reporter gene due to the presence of amutation or a deletion, wherein said desired DNA target is inserted in(i) or (ii) at the place of the deletion or in the vicinity of themutation, and wherein the isolated yeast or mammalian cell furthercomprises a genetic construct comprising the segment of thepolynucleotide encoding the positive selection marker which has beendeleted or mutated, or the segment of the reporter gene which has beendeleted or mutated, flanked at each side by the fragments of thepolynucleotide encoding the positive selection marker or the reportergene bordering the deletion/insertion.
 56. The method according to claim41, wherein the double-strand break in the desired DNA target isrepaired by homologous recombination between two direct repeats.
 57. Themethod according to claim 41, wherein the double-strand break in thedesired DNA target is repaired by gene conversion.
 58. The methodaccording to claim 53, wherein the expression cassette is inserted in aplasmid which is maintained in an episomal form in said isolated yeastor mammalian cell.
 59. The method according to claim 53, wherein theexpression cassette is integrated in the chromosome of said isolatedyeast or mammalian cell.
 60. The method according to claim 41, whereinsaid positive selection marker is an antibiotic resistance or anauxotrophy marker.
 61. The method according to claim 53, wherein saidexpression cassette comprises a additional selection marker.
 62. Amethod for producing LAGLIDADG meganuclease variants derived from aninitial LAGLIDADG meganuclease, said meganuclease variants being able tocleave a desired DNA target which is different from the DNA target ofthe initial LAGLIDADG meganuclease, wherein said method comprises: a)generating a combinatorial library of genes coding for LAGLIDADGmeganuclease variants by introducing diversity into several codons of agene encoding said initial LAGLIDADG meganuclease, wherein said codonscode for amino acid residues of the four-stranded beta-sheet of onedomain or one subunit of said initial LAGLIDADG meganuclease thatinteract directly or indirectly with different bases of the DNA targetof said initial meganuclease; b) selecting meganuclease variants encodedby the genes of the combinatorial library that are able to cleave saiddesired DNA target, by an in vivo cleavage assay in an isolated yeastcell or an isolated mammalian cell, under conditions where adouble-strand break in the desired DNA target which is generated by saidvariant leads to the activation of a positive selection marker, or theinactivation of a negative selection marker, by recombination-mediatedrepair of said desired DNA double-strand break, in the isolated yeastcell or the isolated mammalian cell, and recovering a subset of genesencoding the meganuclease variants able to cleave said desired DNAtarget; c) screening meganuclease variants encoded by the subset ofgenes obtained in b) by an in vivo cleavage assay in an isolated yeastcell or an isolated mammalian cell, under conditions where adouble-strand break in the desired DNA target which is generated by saidvariant leads to the activation of a positive selection marker or areporter gene, or the inactivation of a negative selection marker or areporter gene, by recombination-mediated repair of said desired DNAdouble-strand break, and recovering the subset of meganuclease variantsable to cleave said desired DNA target; d) isolating the genes encodingthe meganuclease variants obtained in c) and able to cleave said desiredDNA target.
 63. The method according to claim 62, wherein said aminoacid residues that interact directly or indirectly with the DNA targetof the initial meganuclease are selected from the group consisting of:D, E, H, K, N, Q, R, S, T and Y.
 64. The method according to claim 63,wherein said amino acid residues are substituted with an amino acidselected from the group consisting of: A, D, E, G, H, K, N, P, Q, S andT.
 65. The method according to claim 62, wherein said initial LAGLIDADGmeganuclease is a natural or a modified meganuclease.
 66. The methodaccording to claim 62, wherein said initial LAGLIDADG meganuclease isselected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, andPI-Pfu I.
 67. The method according to claim 66, wherein said initialLAGLIDADG meganuclease is I-Cre I.
 68. The method according to claim 67,wherein I-Cre I variants are prepared by introducing amino acidsubstitutions in at least three positions of the I-CreI of SEQ ID NO:10, wherein said positions are selected from the group consisting ofQ26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10.
 69. Themethod according to claim 68, wherein said I-Cre I variants compriseamino acid substitutions at the following positions Q26, K28, N30, Y33,Q40, Q44, R68 and R70.
 70. The method according to claim 68, whereinsaid I-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q44, R68 and R70.
 71. The method accordingto claim 68, wherein said I-Cre I variants comprise amino acidsubstitutions at the following positions Q26, K28, Y33, Q38, Q44, R68and R70.
 72. The method according to claim 68, further introducing anamino acid substitution with an uncharged amino acid, at position 75 ofthe I-Cre I of SEQ ID NO:
 10. 73. The method according to claim 72,wherein said uncharged amino acid is an asparagine residue.
 74. Themethod according to claim 62, wherein in said in vivo cleavage assay,said isolated yeast or mammalian cell comprise an expression cassettecomprising said desired DNA target and the coding region of an activenegative selection marker, the coding region of an inactive positiveselection marker, or the coding region of an active or inactive reportergene.
 75. The method according to claim 62, wherein in said in vivocleavage assay, said isolated yeast or mammalian cell comprises anexpression cassette comprising (i) said desired DNA target and apolynucleotide comprising two direct repeats of an internal portion of apolynucleotide encoding an inactive positive selection marker, whereinthe two repeats are separated by the desired DNA target, or (ii) saiddesired DNA target and a polynucleotide comprising two direct repeats ofan internal portion of an inactive reporter gene, wherein the tworepeats are separated by the desired DNA target.
 76. The methodaccording to claim 74, wherein in said in vivo cleavage assay, saidisolated yeast or mammalian cell comprise an expression cassettecomprising said desired DNA target and (i) a polynucleotide encoding aninactive positive selection marker due to the presence of a mutation ora deletion, or (ii) an inactive reporter gene due to the presence of amutation or a deletion, wherein said desired DNA target is inserted in(i) or (ii) at the place of the deletion or in the vicinity of themutation, and wherein the isolated yeast or mammalian cell furthercomprises a genetic construct comprising the segment of thepolynucleotide encoding the positive selection marker which has beendeleted or mutated, or the segment of the reporter gene which has beendeleted or mutated, flanked at each side by the fragments of thepolynucleotide encoding the positive selection marker or the reportergene bordering the deletion/insertion.
 77. The method according to claim62, wherein the double-strand break in the desired DNA target isrepaired by homologous recombination between two direct repeats.
 78. Themethod according to claim 62, wherein the double-strand break in the DNAtarget is repaired by gene conversion.
 79. The method according to claim74, wherein the expression cassette is inserted in a plasmid which ismaintained in an episomal form in said isolated yeast or mammalian cell.80. The method according to claim 74, wherein the expression cassette isintegrated in the chromosome of said isolated yeast or mammalian cell.81. The method according to claim 62, wherein said positive selectionmarker is an antibiotic resistance or an auxotrophy marker.
 82. Themethod according to claim 74, wherein said expression cassette comprisesan additional selection marker.
 83. A method for producing LAGLIDADGmeganuclease variants derived from an initial LAGLIDADG meganuclease,said meganuclease variants being able to cleave a desired DNA targetwhich is different from the DNA target of the initial LAGLIDADGmeganuclease, wherein said method comprises: a) generating acombinatorial library of genes coding for LAGLIDADG meganucleasevariants by introducing diversity into several codons of a gene encodingsaid initial LAGLIDADG meganuclease, wherein said codons code for aminoacid residues of the four-stranded beta-sheet of one domain or onesubunit of said initial meganuclease that interact directly orindirectly with different bases of the DNA target of said initialmeganuclease; b) selecting meganuclease variants encoded by the genes ofthe combinatorial library that are able to bind said desired DNA targetin vitro, under conditions excluding the presence of manganese ormagnesium by incubating said meganuclease variants with said DNA targetimmobilized on a support and recovering only the subset of boundmeganuclease variants; c) screening the subset of bound meganucleasevariants obtained in b) by a binding assay consisting of incubation ofsaid selected meganuclease variants with said desired DNA targetimmobilized on a support and recovering a subset of bound meganucleasevariants; d) selecting among the meganuclease variants recovered in stepc), the variants able to cleave said desired DNA target, by an in vivocleavage assay, in an isolated yeast cell or an isolated mammalian cell,under conditions where the double-strand break in the desired DNA targetwhich is generated by said variant leads to the activation of a positiveselection marker, or the inactivation of a negative selection marker, byrecombination-mediated repair of said DNA double-strand break, andrecovering a subset of meganuclease variants able to cleave said desiredDNA target; e) screening said subset of meganuclease variants obtainedin d) by an in vivo cleavage assay in an isolated yeast cell or amammalian cell, under conditions where the a double-strand break in thedesired DNA target which is generated by said variant leads to theactivation of a positive selection marker or a reporter gene, or theinactivation of a negative selection marker or a reporter gene, byrecombination-mediated repair of said DNA double-strand break, andrecovering a subset of meganuclease variants able to cleave said desiredDNA target; f) isolating the genes encoding the meganuclease variantsobtained in e) which are able to cleave said desired DNA target.
 84. Themethod according to claim 83, wherein said selection and screening stepsuse the phage display method.
 85. The method according to claim 83,wherein said amino acid residues that interact directly or indirectlywith the DNA target of the initial meganuclease are selected from thegroup consisting of: D, E, H, K, N, Q, R, S, T and Y.
 86. The methodaccording to claim 85, wherein said amino acid residues are substitutedwith an amino acid selected from the group consisting of: A, D, E, G, H,K, N, P, Q, S and T.
 87. The method according to claim 83, wherein saidinitial LAGLIDADG meganuclease is a natural or a modified meganuclease.88. The method according to claim 83, wherein said initial LAGLIDADGmeganuclease is selected from the group consisting of: I-Cre I, I-Dmo I,PI-Sce I, and PI-Pfu I.
 89. The method according to claim 88, whereinsaid initial LAGLIDADG meganuclease is I-Cre I.
 90. The method accordingto claim 89, wherein I-Cre I variants are prepared by introducing aminoacid substitutions in at least three positions of the I-CreI of SEQ IDNO: 10, wherein said positions are selected from the group consisting ofQ26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10.
 91. Themethod according to claim 90, wherein said I-Cre I variants compriseamino acid substitutions at the following positions Q26, K28, N30, Y33,Q38, Q44, R68 and R70.
 92. The method according to claim 90, whereinsaid I-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q44, R68 and R70.
 93. The method accordingto claim 90, wherein said I-Cre I variants comprise amino acidsubstitutions at the following positions Q26, K28, Y33, Q38, Q44, R68and R70.
 94. The method according to claim 90, further introducing anamino acid substitution with an uncharged amino acid, at position 75 ofthe I-Cre I of SEQ ID NO:
 10. 95. The method according to claim 64,wherein said uncharged amino acid is an asparagine residue.
 96. Themethod according to claim 83, wherein in said in vivo cleavage assay,said isolated yeast or mammalian cell comprises an expression cassettecomprising said desired DNA target and the coding region of an activenegative selection marker, the coding region of an inactive positiveselection marker, or the coding region of an active or inactive reportergene product.
 97. The method according to claim 83, wherein in said invivo cleavage assay, said isolated yeast or mammalian cell comprises anexpression cassette comprising (i) said desired DNA target and apolynucleotide comprising two direct repeats of an internal portion of apolynucleotide encoding an inactive positive selection marker, whereinthe two repeats are separated by the desired DNA target, or (ii) saiddesired DNA target and a polynucleotide comprising two direct repeats ofan internal portion of an inactive reporter gene, wherein the tworepeats are separated by the desired DNA target.
 98. The methodaccording to claim 96, wherein in said in vivo cleavage assay, saidisolated yeast or mammalian cell comprise an expression cassettecomprising said desired DNA target and (i) a polynucleotide encoding aninactive positive selection marker due to the presence of a mutation ora deletion, or (ii) an inactive reporter gene due to the presence of amutation or a deletion, wherein said desired DNA target is inserted in(i) or (ii) at the place of the deletion or in the vicinity of themutation, and wherein the isolated yeast or mammalian cell furthercomprises a genetic construct comprising the segment of thepolynucleotide encoding the positive selection marker which has beendeleted or mutated, or the segment of the reporter gene which has beendeleted or mutated, flanked at each side by the fragments of thepolynucleotide encoding the positive selection marker or the reportergene bordering the deletion/insertion.
 99. The method according to claim83, wherein the double-strand break in the desired DNA target isrepaired by homologous recombination between two direct repeats. 100.The method according to claim 83, wherein the double-strand break in thedesired DNA target is repaired by gene conversion.
 101. The methodaccording to claim 96, wherein the expression cassette is inserted in aplasmid which is maintained in an episomal form in said isolated yeastor mammalian cell.
 102. The method according to claim 96, wherein theexpression cassette is integrated in the chromosome of said isolatedyeast or mammalian cell.
 103. The method according to claim 83, whereinsaid positive selection marker is an antibiotic resistance or anauxotrophy marker.
 104. The method according to claim 96, wherein saidexpression cassette comprises an additional selection marker.
 105. Amethod for producing LAGLIDADG meganuclease variants derived from aninitial LAGLIDADG meganuclease, said meganuclease variants being able tocleave a desired DNA target which is different from the DNA target ofthe initial LAGLIDADG meganuclease, wherein said method comprises: a)generating a combinatorial library of genes coding for LAGLIDADGmeganuclease variants by introducing diversity into several codons of agene encoding said initial LAGLIDADG meganuclease, wherein said codonscode for amino acid residues of the four-stranded beta-sheet of onedomain or one subunit of said initial LAGLIDADG meganuclease thatinteract directly or indirectly with different bases of the DNA targetof said initial meganuclease; b) selecting meganuclease variants encodedby the genes of the combinatorial library that are able to bind saiddesired DNA target in vitro, under conditions excluding the presence ofmanganese or magnesium by incubating said library with said immobilizeddesired DNA target and recovering only the subset of bound meganucleasevariants; c) screening the subset of bound meganuclease variantsobtained in b) by a binding assay consisting of incubation of saidselected meganuclease variants with said desired DNA target immobilizedon a support and recovering a subset of bound meganuclease variants; d)screening the genes encoding the subset of bound meganuclease variantsobtained in c) by an in vivo cleavage assay in an isolated yeast cell oran isolated mammalian cell, under conditions where the a double-strandbreak in the desired DNA target which is generated by said variant leadsto the activation of a positive selection marker or a reporter gene, orthe inactivation of a negative selection marker or a reporter gene, byrecombination-mediated repair of said DNA double-strand break, and e)isolating the genes encoding the meganuclease variants obtained in d)which are able to cleave said DNA target.
 106. The method according toclaim 105, wherein said selection and screening steps use the phagedisplay method.
 107. The method according to claim 105, wherein saidamino acid residues that interact directly or indirectly with the DNAtarget of the initial meganuclease are selected from the groupconsisting of: D, E, H, K, N, Q, R, S, T and Y.
 108. The methodaccording to claim 107, wherein said amino acid residues are substitutedwith an amino acid selected from the group consisting of: A, D, E, G, H,K, N, P, Q, S and T.
 109. The method according to claim 105, whereinsaid initial LAGLIDADG meganuclease is a natural or a modifiedmeganuclease.
 110. The method according to claim 105, wherein saidinitial LAGLIDADG meganuclease is selected from the group consisting of:I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I.
 111. The method according toclaim 110, wherein said initial LAGLIDADG meganuclease is I-Cre I. 112.The method according to claim 111, wherein I-Cre I variants are preparedby introducing amino acid substitutions in at least three positions ofthe I-CreI of SEQ ID NO: 10, wherein said positions are selected fromthe group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70of SEQ ID NO:10.
 113. The method according to claim 112, wherein saidI-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q38, Q44, R68 and R70.
 114. The methodaccording to claim 112, wherein said I-Cre I variants comprise aminoacid substitutions at the following positions Q26, K28, N30, Y33, Q44,R68 and R70.
 115. The method according to claim 112, wherein said I-CreI variants comprise amino acid substitutions at the following positionsQ26, K28, Y33, Q38, Q44, R68 and R70.
 116. The method according to claim112, further introducing an amino acid substitution with an unchargedamino acid, at position 75 of the I-Cre I of SEQ ID NO:
 10. 117. Themethod according to claim 116, wherein said uncharged amino acid is anasparagine residue.
 118. The method according to claim 105, wherein insaid in vivo cleavage assay, said isolated yeast or mammalian cellcomprises an expression cassette comprising said desired DNA target andthe coding region of an active negative selection marker, the codingregion of an inactive positive selection marker, or the coding region ofan active or inactive reporter gene product.
 119. The method accordingto claim 105, wherein in said in vivo cleavage assay, said isolatedyeast or mammalian cell comprises an expression cassette comprising (i)said desired DNA target and a polynucleotide comprising two directrepeats of an internal portion of a polynucleotide encoding an inactivepositive selection marker, wherein the two repeats are separated by thedesired DNA target, or (ii) said desired DNA target and a polynucleotidecomprising two direct repeats of an internal portion of an inactivereporter gene, wherein the two repeats are separated by the desired DNAtarget.
 120. The method according to claim 118, wherein in said in vivocleavage assay, said isolated yeast or mammalian cell comprise anexpression cassette comprising said desired DNA target and (i) apolynucleotide encoding an inactive positive selection marker due to thepresence of a mutation or a deletion, or (ii) an inactive reporter genedue to the presence of a mutation or a deletion, wherein said desiredDNA target is inserted in (i) or (ii) at the place of the deletion or inthe vicinity of the mutation, and wherein the isolated yeast ormammalian cell further comprises a genetic construct comprising thesegment of the polynucleotide encoding the positive selection markerwhich has been deleted or mutated, or the segment of the reporter genewhich has been deleted or mutated, flanked at each side by the fragmentsof the polynucleotide encoding the positive selection marker or thereporter gene bordering the deletion/insertion.
 121. The methodaccording to claim 105, wherein the double-strand break in the desiredDNA target is repaired by homologous recombination between two directrepeats.
 122. The method according to claim 105, wherein thedouble-strand break in the desired DNA target is repaired by geneconversion.
 123. The method according to claim 118, wherein theexpression cassette is inserted in a plasmid which is maintained in anepisomal form in said isolated yeast or mammalian cell.
 124. The methodaccording to claim 118, wherein the expression cassette is integrated inthe chromosome of said isolated yeast or mammalian cell.
 125. The methodaccording to claim 105, wherein said positive selection marker is anantibiotic resistance or an auxotrophy marker.
 126. The method accordingto claim 118, wherein said expression cassette comprises an additionalselection marker.
 127. A method for producing LAGLIDADG meganucleasevariants derived from an initial LAGLIDADG meganuclease, saidmeganuclease variants being able to cleave a desired DNA target which isdifferent from the DNA target of the initial LAGLIDADG meganuclease,wherein said method comprises: a) generating a combinatorial library ofgenes coding for LAGLIDADG meganuclease variants by introducingdiversity into several codons of a gene encoding said initial LAGLIDADGmeganuclease, wherein said codons code for amino acid residues of thefour-stranded beta-sheet of one domain or one subunit of said initialLAGLIDAG meganuclease that interact directly or indirectly withdifferent bases of the DNA target of said initial meganuclease; b)selecting meganuclease variants encoded by the genes of thecombinatorial library that are able to bind said desired DNA target invitro, under conditions excluding the presence of manganese or magnesiumby incubating said library with said immobilized desired DNA target andrecovering only the subset of bound meganuclease variants; c) selectingamong said subset of bound meganuclease variants obtained in b), themeganuclease variants able to cleave said desired DNA target, by an invivo cleavage assay, in an isolated yeast cell or an isolated mammaliancell, under conditions where the double-strand break in the desired DNAtarget which is generated by said variant leads to the activation of apositive selection marker, or the inactivation of a negative selectionmarker, by recombination-mediated repair of said DNA double-strandbreak, and recovering the subset of meganuclease variants able to cleavesaid desired DNA target; d) screening said subset of meganucleasevariants obtained in step c) by an in vivo cleavage assay in an isolatedyeast cell or an isolated mammalian cell, under conditions where adouble-strand break in the desired DNA target which is generated by saidvariant leads to the activation of a positive selection marker or areporter gene, or the inactivation of a negative selection marker or areporter gene, by recombination-mediated repair of said DNAdouble-strand break, and e) isolating the meganuclease variants obtainedin d) which are able to cleave said desired DNA target.
 128. The methodaccording to claim 127, wherein said selection and screening steps usethe phage display method.
 129. The method according to claim 127,wherein said amino acid residues that interact directly or indirectlywith the DNA target of the initial meganuclease are selected from thegroup consisting of: D, E, H, K, N, Q, R, S, T and Y.
 130. The methodaccording to claim 129, wherein said amino acid residues are substitutedwith an amino acid selected from the group consisting of: A, D, E, G, H,K, N, P, Q, S and T.
 131. The method according to claim 127, whereinsaid initial LAGLIDADG meganuclease is a natural or a modifiedmeganuclease.
 132. The method according to claim 127, wherein saidinitial LAGLIDADG meganuclease is selected from the group consisting of:I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I.
 133. The method according toclaim 132, wherein said initial LAGLIDADG meganuclease is I-Cre I. 134.The method according to claim 133, wherein I-Cre I variants are preparedby introducing amino acid substitutions in at least three positions ofthe I-CreI of SEQ ID NO: 10, wherein said positions are selected fromthe group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70of SEQ ID NO:10.
 135. The method according to claim 134, wherein saidI-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q38, Q44, R68 and R70.
 136. The methodaccording to claim 134, wherein said I-Cre I variants comprise aminoacid substitutions at the following positions Q26, K28, N30, Y33, Q44,R68 and R70.
 137. The method according to claim 134, wherein said I-CreI variants comprise amino acid substitutions at the following positionsQ26, K28, Y33, Q38, Q44, R68 and R70.
 138. The method according to claim134, further introducing an amino acid substitution with an unchargedamino acid, at position 75 of the I-Cre I of SEQ ID NO:
 10. 139. Themethod according to claim 138, wherein said uncharged amino acid is anasparagine residue.
 140. The method according to claim 127, wherein insaid in vivo cleavage assay, said isolated yeast or mammalian cellcomprises an expression cassette comprising said desired DNA target andthe coding region of an active negative selection marker, the codingregion of an inactive positive selection marker, or the coding region ofan active or inactive reporter gene.
 141. The method according to claim127, wherein in said in vivo cleavage assay, said isolated yeast ormammalian cell comprises an expression cassette comprising (i) saiddesired DNA target and a polynucleotide comprising two direct repeats ofan internal portion of a polynucleotide encoding an inactive positiveselection marker, wherein the two repeats are separated by the desiredDNA target, or (ii) said desired DNA target and a polynucleotidecomprising two direct repeats of an internal portion of an inactivereporter gene, wherein the two repeats are separated by the desired DNAtarget.
 142. The method according to claim 140, wherein in said in vivocleavage assay, said isolated yeast or mammalian cell comprise anexpression cassette comprising said desired DNA target and (i) apolynucleotide encoding an inactive positive selection marker due to thepresence of a mutation or a deletion, or (ii) an inactive reporter genedue to the presence of a mutation or a deletion, wherein said desiredDNA target is inserted in (i) or (ii) at the place of the deletion or inthe vicinity of the mutation, and wherein the isolated yeast ormammalian cell further comprises a genetic construct comprising thesegment of the polynucleotide encoding the positive selection markerwhich has been deleted or mutated, or the segment of the reporter genewhich has been deleted or mutated, flanked at each side by the fragmentsof the polynucleotide encoding the positive selection marker or thereporter gene bordering the deletion/insertion.
 143. The methodaccording to claim 127, wherein the double-strand break in the desiredDNA target is repaired by homologous recombination between two directrepeats.
 144. The method according to claim 127, wherein thedouble-strand break in the desired DNA target is repaired by geneconversion.
 145. The method according to claim 140, wherein theexpression cassette is inserted in a plasmid which is maintained in anepisomal form in said isolated yeast or mammalian cell.
 146. The methodaccording to claim 114, wherein the expression cassette is integrated inthe chromosome of said isolated yeast or mammalian cell.
 147. The methodaccording to claim 127, wherein said positive selection marker is anantibiotic resistance or an auxotrophy marker.
 148. The method accordingto claim 140, wherein said expression cassette comprises an additionalselection marker.
 149. A method for producing LAGLIDADG meganucleasevariants derived from an initial LAGLIDADG meganuclease, saidmeganuclease variants being able to cleave a desired DNA target which isdifferent from the DNA target of the initial LAGLIDADG meganuclease,wherein said method comprises: a) generating a combinatorial library ofgenes coding for LAGLIDADG meganuclease variants by introducingdiversity into several codons of a gene encoding said initial LAGLIDADGmeganuclease, wherein said codons code for amino acid residues of thefour-stranded beta-sheet of one domain or one subunit of said initialLAGLIDADG meganuclease that interact directly or indirectly withdifferent bases of the DNA target of said initial meganuclease; b)screening said meganuclease variants encoded by the genes of thecombinatorial library obtained in a) by a binding assay consisting ofincubating said meganuclease variants encoded by the genes of thecombinatorial library with said desired DNA target immobilized on asupport and recovering a subset of meganuclease variants that are boundto the desired DNA target; c) screening said subset of meganucleasevariants obtained in b) by an in vivo cleavage assay in an isolatedyeast cell or an isolated mammalian cell, under conditions where thedouble-strand break in the desired DNA target which is generated by saidvariant leads to the activation of a positive selection marker or areporter gene, or the inactivation of a negative selection marker or areporter gene, by recombination-mediated repair of said DNAdouble-strand break, and d) isolating the meganuclease variants obtainedin c) which are able to cleave said desired DNA target.
 150. The methodaccording to claim 149, wherein said selection and screening steps usethe phage display method.
 151. The method according to claim 149,wherein said amino acid residues that interact directly or indirectlywith the DNA target of the initial meganuclease are selected from thegroup consisting of: D, E, H, K, N, Q, R, S, T and Y.
 152. The methodaccording to claim 149, wherein said amino acid residues are substitutedwith an amino acid selected from the group consisting of: A, D, E, G, H,K, N, P, Q, S and T.
 153. The method according to claim 149, whereinsaid initial LAGLIDADG meganuclease is a natural or a modifiedmeganuclease.
 154. The method according to claim 149, wherein saidinitial LAGLIDADG meganuclease is selected from the group consisting of:I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I.
 155. The method according toclaim 154, wherein said initial LAGLIDADG meganuclease is I-Cre I. 156.The method according to claim 155, wherein I-Cre I variants are preparedby introducing amino acid substitutions in at least three positions ofthe I-CreI of SEQ ID NO: 10, wherein said positions are selected fromthe group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70of SEQ ID NO:10.
 157. The method according to claim 156, wherein saidI-Cre I variants comprise amino acid substitutions at the followingpositions Q26, K28, N30, Y33, Q38, Q44, R68 and R70.
 158. The methodaccording to claim 156, wherein said I-Cre I variants comprise aminoacid substitutions at the following positions Q26, K28, N30, Y33, Q44,R68 and R70.
 159. The method according to claim 156, wherein said I-CreI variants comprise amino acid substitutions at the following positionsQ26, K28, Y33, Q38, Q44, R68 and R70.
 160. The method according to claim156, further introducing an amino acid substitution with an unchargedamino acid, at position 75 of the I-Cre I of SEQ ID NO:
 10. 161. Themethod according to claim 160, wherein said uncharged amino acid is anasparagine residue.
 162. The method according to claim 149, wherein insaid in vivo cleavage assay, said isolated yeast or mammalian cellcomprises an expression cassette comprising said desired DNA target andthe coding region of an active negative selection marker, the codingregion of an inactive positive selection marker, or the coding region ofan active or inactive reporter gene.
 163. The method according to claim149, wherein in said in vivo cleavage assay, said isolated yeast ormammalian cell comprises an expression cassette comprising (i) saiddesired DNA target and a polynucleotide comprising two direct repeats ofan internal portion of a polynucleotide encoding an inactive positiveselection marker, wherein the two repeats are separated by the desiredDNA target, or (ii) said desired DNA target and a polynucleotidecomprising two direct repeats of an internal portion of an inactivereporter gene, wherein the two repeats are separated by the desired DNAtarget.
 164. The method according to claim 162, wherein in said in vivocleavage assay, said isolated yeast or mammalian cell comprise anexpression cassette comprising said desired DNA target and (i) apolynucleotide encoding an inactive positive selection marker due to thepresence of a mutation or a deletion, or (ii) an inactive reporter genedue to the presence of a mutation or a deletion, wherein said desiredDNA target is inserted in (i) or (ii) at the place of the deletion or inthe vicinity of the mutation, and wherein the isolated yeast ormammalian cell further comprises a genetic construct comprising thesegment of the polynucleotide encoding the positive selection markerwhich has been deleted or mutated, or the segment of the reporter genewhich has been deleted or mutated, flanked at each side by the fragmentsof the polynucleotide encoding the positive selection marker or thereporter gene bordering the deletion/insertion.
 165. The methodaccording to claim 149, wherein the double-strand break in the desiredDNA target is repaired by homologous recombination between two directrepeats.
 166. The method according to claim 149, wherein thedouble-strand break in the desired DNA target is repaired by geneconversion.
 167. The method according to claim 162, wherein theexpression cassette is inserted in a plasmid which is maintained in anepisomal form in said isolated yeast or mammalian cell.
 168. The methodaccording to claim 162, wherein the expression cassette is integrated inthe chromosome of said isolated yeast or mammalian cell.
 169. The methodaccording to claim 149, wherein said positive selection marker is anantibiotic resistance or an auxotrophy marker.
 170. The method accordingto claim 162, wherein said expression cassette comprises an additionalselection marker.